Reshaped human anti-hm 1.24 antibody

ABSTRACT

A reshaped human anti-HM 1.24 antibody comprising:  
     (A) an L chain comprising  
     (1) the C region of a human L chain, and  
     (2) the V region of an L chain comprising the FR of a human L chain and the CDR of the L chain of a mouse anti-HM 1.24 monoclonal antibody; and  
     (B) an H chain comprising  
     (1) the C region of a human H chain, and  
     (2) the V region of an H chain comprising the FR (of a human H chain and the CDR of the H chain of a mouse anti-HM 1.24 monoclonal antibody.  
     Since most of this reshaped human antibody is derived from human antibody and the CDR has a low antigenicity, the reshaped human antibody of the present invention has a low antigenicity and, therefore, is expected to be used for medical treatment.

FIELD OF THE INVENTION

[0001] The present invention relates to reshaped human anti-HM 1.24antibodies and chimeric anti-HM 1.24 antibodies, genes encoding them,methods for producing said antibodies, and the use of said antibodies.The reshaped human antibodies and the chimeric antibodies of the presentinvention are useful as a therapeutic agent, etc. for myeloma.

BACKGROUND ART

[0002] Human B cells go through a variety of processes that areclassified based on the kind of surface antigens being expressed, andfinally mature into antibody-producing plasma cells. At the final stageof their differentiation, B cells, on one hand, acquire the ability ofproducing cytoplasmic immunoglobulins and, on the other, Bcell-associated antigens such as cell surface immunoglobulins, HLA-DR,CD20, Fc receptors, complement C3 receptors and the like disappear(Ling, N.R. et al., Leucocyte Typing III (1986) p320, Oxford, UK,Oxford).

[0003] So far, there have been reports on monoclonal antiboies such asanti-PCA-1 (Anderson, K.C. et al., J. Immunol. (1983) 130, 1132),anti-PC-1 (Anderson, K. C. et al., J. Immunol. (1983) 132, 3172),anti-MM4 (Tong, A. W. et al., Blood (1987) 69, 238) and the like thatrecognize antigens on the cell membrane of the plasma cells. However,anti-CD38 monoclonal antibody is still being used for detection ofplasma cells and myeloma cells (Epstein, J. et al., N. Engl. J. Med.(1990) 322, 664, Terstappen, L.W.M.M. et al., Blood (1990) 76, 1739,Leo, R. et al., Ann. Hematol. (1992) 64, 132, Shimazaki, C. et al., AmJ. Hematol. (1992) 39, 159, Hata, H. et al., Blood (1993) 81, 3357,Harada, H. et al., Blood (1993) 81, 2658, Billadeau, D. et al., J. Exp.Med. (1993) 178, 1023).

[0004] However, anti-CD38 monoclonal antibody is an antigen associatedwith activation of T cells rather than an antigen associated withdifferentiation of B cells, and is expressed on various cells inaddition to B cells. Furthermore, although CD38 is not expressed on someof the lymphoplasmacytoid, it is strongly expressed on the hemopoieticprecursor cells. For these reasons, it is believed that anti-CD38monoclonal antibody is not suitable for research on differentiation andmaturation of human B cells or for treatment of diseases of plasmacells.

[0005] Goto, T. et al. have reported mouse anti-HM 1.24 monoclonalantibody that recognizes an antigen having a molecular weight of 29 to33 kDa which is specifically expressed on B cell lines (Blood (1994) 84,1922-1930). From the fact that the antigen recognized by anti-HM 1.24monoclonal antibody is believed to be associated with the terminaldifferentiation of B cells (Goto, T. et al., Jpn. J. Clin. Immun. (1992)16, 688-691) and that the administration of anti-HM 1.24 monoclonalantibody to a plasmacytoma-transplanted mouse resulted in specificaccumulation of the antibody at the tumor (Shuji Ozaki et al., TheProgram of General Assembly of the 19th Japan Myeloma Study Meeting,general presentation 3), it has been suggested that anti-HM 1.24monoclonal antibody, by labelling with a radioisotope, may be used fordiagnosis of tumor localization, the missile therapy such asradioimmunotherapy, and the like.

[0006] Furthermore, the above-mentioned Blood describes that the anti-HM1.24 monoclonal antibody has the complement-dependent cytotoxicityactivity to the human myeloma cell line RPMI8226.

[0007] Myeloma is a neoplastic disease characterized by the accumulationof monoclonal plasma cells (myeloma cells) in the bone marrow. Myelomais a disease in which terminally differentiated B cells that produce andsecrete immunoglobulins, or plasma cells, are monoclonally increasedmainly in the bone marrow, and accordingly monoclonal immunoglobulins orthe constituting components thereof, L chains or H chains, are detectedin the serum (Masaaki Kosaka et al., Nippon Rinsho (1995) 53, 91-99).

[0008] Conventionally chemotherapeutic agents have been used fortreatment, of myeloma, but there have been found no effectivetherapeutic agents that can lead to remission of myeloma and elongationof the survival period of patients with myeloma. There is, therefore, along-awaited need for the advent of drugs that have a therapeutic effecton myeloma.

[0009] Mouse monoclonal antiboies have high immunogenicity (sometimesreferred to as “antigenicity”) in humans. Accordingly, the medicaltherapeutic value of mouse monoclonal antibodies in humans is limited.For example, a mouse antibody administered into a human may bemetabolized as a foreign substance so that the half life of the mouseantibody in the human is relatively short and thereby it cannot fullyexhibit its expected effects. Furthermore, human anti-mouse antibodiesthat are raised against the administered mouse antibody may triggerimmunological responses that are unfavorable and dangerous to thepatients, such as serum disease, other allergic reactions, or the like.Therefore, mouse monoclonal antibody cannot be frequently administeredinto humans.

[0010] In order to resolve these problems, a method was developed forreducing the immunogenicity of non-human-derived antibodies such asmouse-derived monoclonal antibodies. As one such example, there is amethod of producing a chimeric antibody in which the variable region (vregion) of the antibody is derived from the original mouse and theconstant region (C region) thereof is derived from an appropriate humanantibody.

[0011] Since the chimeric antibody thus obtained contains the variableregion of the original mouse antibody in the intact form, it is expectedto bind to the antigen with a specificity identical to that of theoriginal mouse antibody. Furthermore, in a chimeric antibody the ratioof the amino acid sequences derived from non-humans is substantiallyreduced, and so the antibody is expected to have a low immunogenicitycompared to the original mouse antibody. A chimeric antibody may bind tothe antigen in an equal manner to the original mouse monoclonalantibody, and may include immunological responses against the mousevariable region though the immunogenicity is reduced (LoBuglio, A. F. etal., Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989).

[0012] The second method for reducing the immunogenicity of mouseantibody, though much more complicated, can reduce the potentialimmunogenicity of mouse antibody further greatly. In this method, onlythe complementarity determining region (CDR) of the variable region of amouse antibody is grafted to the variable region of a human antibody toprepare a “reshaped” human antibody variable region.

[0013] However, In order to make the structure of the CDR of a reshapedhuman antibody variable region as much close as possible to that of theoriginal mouse antibody, if necessary, part of the amino acid sequenceof the framework region (FR) that supports the CDR may be grafted fromthe variable region of the mouse antibody to the variable region of thehuman antibody. Subsequently, this V region of the humanized reshapedhuman antibody is linked to the constant region of a human antibody. Thepart that is derived from the non-human amino acid sequence in thefinally reshaped humanized antibody is the CDR, and only part of the FR.A CDR is composed of hypervariable amino acid sequences which do notexhibit species-specific sequences. Therefore, the humanized antibodycarrying the mouse CDR should not have an immunogenicity stronger thanthe natural human antibody having the human antibody CDR.

[0014] For the humanized antibody, see Riechmann, L. et al., Nature,332, 323-327, 1988; Verhoeye, M. et al., Science, 239, 1534-1536, 1988;Kettleborough, C. A. et al., Protein Engng., 4, 773-783, 1991; Meada, H.et al., Human Antibodies and Hybridoma, 2, 124-134, 1991; Groman, S.D.et al., Proc. Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest, P. R.et al., Bio/Technology, 9, 266-271; 1991; Co, M. S. et al., Proc. Natl.Acad. Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl.Acad. Sci. USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148,1149-1154, 1992; and Sato, K. et al, Cancer Res., 53, 851-856, 1993.

[0015] Queen et al. (International Application Publication No. WO90-07861) describes a method for producing a humanized antibody of ananti-IL-2 receptor antibody Anti-Tac. However, it is difficult tocompletely humanize all antibodies even following the method as setforth in WO 90-07861. Thus, WO 90-07861 does not describe a generalmethod for humanizing of antibodies, but merely describes a method forhumanizing of Anti-Tac antibody which is one of anti-IL-2 receptorantibodies. Furthermore, even when the method of WO 90-07861 iscompletely followed, it is difficult to make a humanized antibody thathas an activity completely identical to the original mouse antibody.

[0016] In general, the amino acid sequences of CDR/FR of individualantibodies are different. Accordingly, the determination of the aminoacid residue to be replaced for the construction of a humanized antibodyand the selection of the amino acid residue that replaces said aminoacid residue vary with individual antibodies. Therefore, the method forpreparing humanized antibodies as set forth in WO 90-07861 cannot beapplied to humanization of all antibodies.

[0017] Queen et al. Proc. Natl. Acad. Sci. USA, (1989) 86, 10029-10033has a similar disclosure to that of WO 90-07861. This referencedescribes that only one third of the activity of the original mouseantibody was obtained for a humanized antibody produced according to themethod as set forth in WO 90-07861. In other words, this shows that themethod of WO 90-07861 itself cannot produce a complete humanizedantibody that has an activity equal to that of the original mouseantibody Co et al., Cancer Research (1996) 56, 1118-1125 was publishedby the group of the above-mentioned Queen et al. This referencedescribes that a humanized antibody having an activity equal to that ofthe original mouse antibody could not be constructed even by the methodfor making humanized antibody as set forth in WO 90-07861. Thus, thefact not only reveals that the method of WO 90-07861 itself cannotproduce a complete humanized antibody having an activity equal to theoriginal mouse antibody, but that the method for constructing humanizedantibody as set forth in WO 90-07861 cannot be applied to humanizationof all antibodies.

[0018] Ohtomo et al., Molecular Immunology (1995) 32, 407-416 describeshumanization of mouse ONS-M21 antibody. This reference reveals that theamino acid residue which was suggested for humanization of the Anti-Tacantibody in WO 90-07861 has no relation with the activity and the methodas set forth in WO 90-07861 cannot be applied.

[0019] Kettleborough et al, Protein Eng. (1991) 4, 773-783 disclosesthat several humanized antibodies were constructed from mouse antibodyby substituting amino acid residues. However, the substitution of moreamino acid residues than were suggested in the method of humanization ofthe Anti-Tac antibody as described in WO 90-07861 was required.

[0020] The foregoing references indicate that the method of producinghumanized antibodies as set forth in WO 90-07861 is a techniqueapplicable only to the Anti-Tac antibody described therein and that eventhe use of said technology does not lead to the activity equal to thatof the original mouse antibody.

[0021] The original mouse antibodies described in these references havedifferent amino acid sequences from that of the Anti-Tac antibodydescribed in WO 90-07861. Accordingly, the method of constructinghumanized antibody which was able to be applied to the Anti-Tac antibodycould not be applied to other antibodies. Similarly, since the mouseanti-HM 1.24 antibody of the present invention has an amino acidsequence different from that of the Anti-Tac antibody, the method ofconstructing humanized antibody for the Anti-Tac antibody cannot beapplied. Furthermore, the successfully constructed humanized antibody ofthe present invention has an amino acid sequence different from that ofthe humanized Anti-Tac antibody described in WO 90-07861. This fact alsoindicates that the same method cannot be applied for humanization ofantibodies having different CDR-FR sequences.

[0022] Thus, even if the original mouse antibody for humanization isknown, the identity of the CDR-FR sequence of a humanized antibodyhaving an activity is confirmed only after trial and error experiments.WO 90-07861 makes no mention of the FR sequence which is combined in thehumanized antibody constructed in the present invention and of the factthat an active humanized antibody could be obtained from the combinationwith FR, much less the sequence of the CDR.

[0023] As hereinabove mentioned, humanized antibodies are expected to beuseful for therapeutic purposes, but humanized anti-HM 1.24 antibody isnot known or not even suggested. Furthermore, there is no standardizedmethod available that could be generally applied to any antibody forproduction of a humanized antibody, and a variety of contrivances areneeded for constructing a humanized antibody that exhibits sufficientbinding activity, binding inhibition activity, and neutralizing activity(for example, Sato, K. et al., Cancer Res., 53, 851-856, 1993).

[0024] Disclosure of the Invention

[0025] The present invention provides reshaped antibodies of anti-HM1.24 antibody. The present invention further provides human/mousechimeric antibodies that are useful in the process of constructing saidreshaped antibodies. The present invention further provides fragments ofthe reshaped antibodies. Furthermore, the present invention provides anexpression system for production of chimeric antibodies, reshapedantibodies and the fragments thereof. The present invention furtherprovides methods for producing chimeric antibodies of anti-HM 1.24antibody and fragments thereof, as well as reshaped antibodies ofanti-HM 1.24 antibody and fragments thereof.

[0026] More specifically, the present invention provides chimericantibodies and reshaped antibodies that specifically recognize apolypeptide having the amino acid sequence as set forth in SEQ ID NO:103. cDNA that encodes said polypeptide has been inserted between theXbaI cleavage sites of pUC19 vector, and thereby been prepared asplasmid pRS38-pUC19. Escherichia coli that contains this plasmidpRS38-pUC19 has been internationally deposited on Oct. 5,1993, with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukubacity, Ibalaki prefecture, Japan) as Escherichia coli DH5α (pRS38-pUC19)under the accession number FERM BP-4434 under the provisions of theBudapest Treaty (see Japanese Unexamined Patent Publication (Kokai) No.7-196694).

[0027] As one embodiment of such chimeric antibodies or reshapedantibodies, there is mentioned a chimeric anti-HM 1.24 antibody or areshaped human anti-HM 1.24 antibody. A detailed description of achimeric anti-HM 1.24 antibody or a reshaped human anti-HM 1.24 antibodywill be given hereinbelow.

[0028] Thus, the present invention also provides chimeric L chainscomprising the constant region (C region) of a human light (L) chain andthe variable (V) region of the L chain of an anti-HM 1.24 antibody, anda chimeric H chain comprising the constant region of a human heavy (H)chain and the V region of anti-HM 1.24 antibody heavy (H) chain.

[0029] The present invention further provides chimeric antibodiescomprising:

[0030] (1) an L chain comprising the C region of a human L chain and theV region of the L chain of an anti-HM 1.24 antibody; and

[0031] (2) an H chain comprising the C region of a human H chain and theV region of the H chain of an anti-HM 1.24 antibody.

[0032] The present invention further provides the V region of thereshaped human L chain of anti-HM 1.24 antibody comprising:

[0033] (1) the framework region (FR) of the V region of a human L chain,and

[0034] (2) the CDR of the V region of the L chain of an anti-HM 1.24antibody; and

[0035] the V region of the reshaped human H chain of anti-HM 1.24antibody comprising

[0036] (1) the FR of the V region of a human H chain, and

[0037] (2) the CDR of the V region of the H chain of an anti-HM 1.24antibody.

[0038] The present invention further provides

[0039] the reshaped human L chain of anti-HM 1.24 antibody comprising

[0040] (1) the C region of a human L chain, and

[0041] (2) the V region of an L chain comprising the FR of a human Lchain and the CDR of the L chain of an anti-HM 35 1.24 antibody; and

[0042] the reshaped human H chain of anti-HM 1.24 antibody comprising

[0043] (1) the C region of a human H chain, and

[0044] (2) the V region of an H chain comprising the FR of a human Hchain and the CDR of the H chain of an anti-HM 1.24 antibody.

[0045] The present invention further provides the reshaped humanantibody of anti-HM 1.24 antibody comprising:

[0046] (A) an L chain comprising

[0047] (1) the C region of a human L chain, and

[0048] (2) the V region of an L chain comprising the FR of a human Lchain and the CDR of the L chain of an anti-HM 1.24 antibody; and

[0049] (B) an H chain comprising

[0050] (1) the C region of a human H chain, and

[0051] (2) the V region of an H chain comprising the FR of a human Hchain and the CDR of the H chain of an anti-HM 1.24 antibody.

[0052] The present invention further provides DNA encoding the V regionof the L chain of an anti-HM 1.24 antibody, and DNA encoding the Vregion of the H chain of an anti-HM 1.24 antibody.

[0053] The present invention further provides

[0054] DNA encoding a chimeric L chain comprising

[0055] (1) the C region of a human L chain; and

[0056] (2) the V region of the L chain of an anti-HM 1.24 antibody, and

[0057] DNA encoding a chimeric H chain comprising

[0058] (1) the C region of a human H chain; and

[0059] (2) the V region of the H chain of an anti-HM 1.24 antibody.

[0060] The present invention further provides

[0061] DNA encoding the V region of the reshaped human L chain ofanti-HM 1.24 antibody comprising:

[0062] (1) the FR of the V region of a human L chain; and

[0063] (2) the CDR of the V region of the L chain of an anti-HM 1.24antibody; and

[0064] DNA encoding the V region of the reshaped human H chain ofanti-HM 1.24 antibody comprising:

[0065] (1) the FR of the V region of a human H chain; and

[0066] (2) the CDR of the V region of the H chain of an anti-HM 1.24antibody.

[0067] The present invention further provides

[0068] DNA encoding the reshaped human L chain of an anti-HM 1.24antibody comprising:

[0069] (1) the C region of a human L chain; and

[0070] (2) the V region of an L chain comprising the FR of a human Lchain and the CDR of the L chain of an anti-HM 1.24 antibody; and

[0071] DNA encoding the reshaped human H chain of an anti-HM 1.24antibody comprising:

[0072] (1) the C region of a human H chain; and

[0073] (2) the V region of an H chain comprising the FR of a human Hchain and the CDR of the H chain of an anti-HM 1.24 antibody.

[0074] The present invention further provides a vector comprising any ofthe various DNAs mentioned above.

[0075] The present invention further provides a host cell transformedwith the above vector.

[0076] The present invention also provides methods for producing thechimeric antibody of an anti-HM 1.24 antibody comprising the steps ofculturing a host cell which was cotransformed with an expression vectorcomprising DNA encoding said chimeric L chain and an expression vectorcomprising DNA encoding said H chain, and of recovering the desiredantibody.

[0077] The present invention further provides methods for producing thereshaped human antibody of an anti-HM 1.24 antibody comprising the stepsof culturing a host cell which was cotransformed with an expressionvector comprising DNA encoding said reshaped human L chain and anexpression vector comprising DNA encoding said reshaped human H chain,and of recovering the desired antibody.

[0078] The present invention further provides pharmaceuticalcompositions, especially therapeutic agents for myeloma, comprising saidchimeric antibody or the reshaped human antibody.

[0079] The present invention further provides pharmaceuticalcompositions which contain as an active ingredient a chimeric antibodyspecifically recognizing a polypeptide having the amino acid sequence asset forth in SEQ ID NO: 103, and pharmaceutical compositions whichcontain as an active ingredient a reshaped human antibody specificallyrecognizing a polypeptide having the amino acid sequence as set forth inSEQ ID NO: 103. As a pharmaceutical composition, there is specificallyprovided a therapeutic agent for myeloma.

BRIEF EXPLANATION OF THE DRAWINGS

[0080]FIG. 1 is a graph showing that, in the FCM analysis using thehuman myeloma cell line KPMM2, the fluorescence intensity of a chimericanti-HM 1.24 antibody is shifted in a similar manner to that of a mouseanti-HM 1.24 antibody as compared to the control antibody.

[0081]FIG. 2 is a graph showing that, in the Cell-ELISA using the WISHcell, the chimeric anti-HM 1.24 antibody similarly to the mouse anti-HM1.24 antibody inhibits the binding of the biotinylated mouse anti-HM1.24 antibody to the WISH cells in a dose dependent manner.

[0082]FIG. 3 is a graph showing that the control human IgG1 or the mouseanti-HM 1.24 antibody has no cytotoxicity whereas the chimeric anti-HM1.24 antibody exhibits increased cytotoxicity to the RPMI 8226 cell withthe increased ratio of E/T.

[0083]FIG. 4 is a diagramatic representation of a method forconstructing the L chain of a reshaped human anti-HM 1.24 antibody byCDR grafting in the PCR method.

[0084]FIG. 5 is a diagramatic representation of a method for assemblyingthe oligonucleotides of RVH1, RVH2, RVH3, and RVH4 by the PCR method inthe preparation of the H chain of the reshaped human anti-HM 1.24antibody.

[0085]FIG. 6 is a diagramatic representation of a method forconstructing the V region of the H chain of a human mouse hybrid anti-HM1.24 antibody by the PCR method.

[0086]FIG. 7 is a diagramatic representation of a method forconstructing the V region of the H chain of a mouse human hybrid anti-HM1.24 antibody by the PCR method.

[0087]FIG. 8 is a graph showing that the version a of the L chain of areshaped human anti-HM 1.24 antibody has an antigen biding activityequal to that of the chimeric anti-HM 1.24 antibody. -1 and -2 show thatthey are different lots.

[0088]FIG. 9 is a graph showing the antigen binding activity of areshaped human anti-HM 1.24 antibody in which the version a of the Lchain and the version a, b, f, or h of the H chain have been combined,and a chimeric anti-HM 1.24 antibody.

[0089]FIG. 10 is a graph showing the binding activity of a reshapedhuman anti-HM 1.24 antibody in which the version b of the L chain andthe version a, b, f, or h of the H chain have been combined, and achimeric anti-HM 1.24 antibody.

[0090]FIG. 11 is a graph showing the binding inhibition activity of areshaped human anti-HM 1.24 antibody in which the version a of the Lchain and H chain version a, b, f, or h have been combined, and achimeric anti-HM 1.24 antibody.

[0091]FIG. 12 is a graph showing the binding inhibition activity of areshaped human anti-HM 1.24 antibody in which the version b of the Lchain and the version a, b, f, or h of the H chain have been combined,and a chimeric anti-HM 1.24 antibody.

[0092]FIG. 13 is a graph showing the antigen binding activity of theversions a, b, c, and d of the H chain of a reshaped human anti-HM 1.24antibody, and a chimeric anti-HM 1.24 antibody.

[0093]FIG. 14 is a graph showing the antigen binding activity of theversions a and e of the H chain of a reshaped human anti-HM 1.24antibody, and a chimeric anti-HM 1.24 antibody. -1 and -2 show that theyare different lots.

[0094]FIG. 15 is a graph showing the binding inhibition activity of theversions a, c, p, and r of the H chain of a reshaped human anti-HM 1.24antibody, and a chimeric anti-HM 1.24 antibody.

[0095]FIG. 16 is a graph showing the antigen binding activity of a humanmouse hybrid anti-HM 1.24 antibody, a mouse human hybrid anti-HM 1.24antibody, and a chimeric anti-HM 1.24 antibody.

[0096]FIG. 17 is a graph showing the antigen binding activity of theversions a, b, c, and f of the H chain of a reshaped human anti-HM 1.24antibody, and a chimeric anti-HM 1.24 antibody.

[0097]FIG. 18 is a graph showing the antigen binding activity of theversions a and g of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0098]FIG. 19 is a graph showing the binding inhibition activity of theversions a and g of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0099]FIG. 20 is a graph showing the antigen binding activity of theversions h and i of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0100]FIG. 21 is a graph showing the antigen binding activity of theversions f, h, and j of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0101]FIG. 22 is a graph showing the binding inhibition activity of theversions h and i of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0102]FIG. 23 is a graph showing the binding inhibition activity of theversions f, h, and j of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0103]FIG. 24 is a graph showing the antigen binding activity of theversions h, k, l, m, n, and O of the H chain of a reshaped human anti-HM1.24 antibody and a chimeric anti-HM 1.24 antibody.

[0104]FIG. 25 is a graph showing the antigen binding activity of theversions a, h, p, and q of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0105]FIG. 26 is a graph showing the inhibition activity of binding tothe WISH cell of the versions h, k, l, m, n, and o of the H chain of areshaped human anti-HM 1.24 antibody and a chimeric anti-HM 1.24antibody.

[0106]FIG. 27 is a graph showing the binding inhibition activity of theversions a, h, p, and q of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0107]FIG. 28 is a graph showing the antigen binding activity of theversions a, c, p, and r of the H chain of a reshaped human anti-HM 1.24antibody and a chimeric anti-HM 1.24 antibody.

[0108]FIG. 29 is a graph showing that the version s of a reshaped humananti-HM 1.24 antibody has an antigen binding activity equal to that ofthe version r of the reshaped human anti-HM 1.24 antibody.

[0109]FIG. 30 is a graph showing that the version s of a reshaped humananti-HM 1.24 antibody has a binding inhibition activity equal to that ofthe version r of the reshaped human anti-HM 1.24 antibody.

[0110]FIG. 31 is a graph showing that a purified reshaped human anti-HM1.24 antibody has an antigen binding activity equal to that of achimeric anti-HM 1.24 antibody.

[0111]FIG. 32 is a graph showing that a purified reshaped human anti-HM1.24 antibody has a binding inhibition activity equal to that of achimeric anti-HM 1.24 antibody.

[0112]FIG. 33 is a graph showing that the administration of a chimericanti-HM 1.24 antibody caused prolongation of the survival period ascompared to the administration of the control human IgG1 in a humanmyeloma cells-transplanted mouse.

[0113]FIG. 34 is a graph showing that when cells derived from theperipheral blood of healthy human are used as a effector cell thecontrol human IgG1 exhibits no cytotoxicity to the KPMM2 cells and amouse anti-HM 1.24 antibody also has a weak cytotoxicity whereas areshaped human anti-HM 1.24 antibody exhibits a strong cytotoxicity tothe KPMM2 cells.

[0114]FIG. 35 is a graph showing that when cells derived from theperipheral blood of healthy human are used as a effector cell thecontrol human IgG1 exhibits no cytotoxicity to the ARH-77 cells and amouse anti-HM 1.24 antibody also has a weak cytotoxicity, whereas areshaped human anti-HM 1.24 antibody exhibits a strong cytotoxicity tothe ARH-77 cells.

[0115]FIG. 36 is a graph showing that when cells derived from the bonemarrow of SCID mice are used as a effector cell the control human IgG1exhibits no cytotoxicity to the KPMM2 cells, whereas a reshaped humananti-HM 1.24 antibody exhibits an increased cytotoxicity to the KPMM2cells with the increase in the antibody concentration.

[0116]FIG. 37 is a graph showing that in a human myelomacells-transplanted mouse the serum IgG human level is increased afterthe administration of the control human IgG1 as compared to the levelbefore the administration, whereas the administration of a reshapedhuman anti-HM 1.24 antibody inhibits the increase in the serum human IgGlevel.

[0117]FIG. 38 is a graph showing that in a human myelomacells-transplanted mouse the administration of a reshaped human anti-HM1.24 antibody causes prolongation of the survival period as compared tothe administration of the control human IgG1.

[0118]FIG. 39 is a graph showing that in a human myelomacells-transplanted mouse the serum human IgG level is increased afterthe administration of melphalan and the control human IgG1 as comparedto the level before the administration, whereas the administration of areshaped human anti-HM 1.24 antibody inhibits the increase in the serumhuman IgG level.

[0119]FIG. 40 is a graph showing that in a human myelomacells-transplanted mouse the administration of a reshaped human anti-HM1.24 antibody causes prolongation of the survival period as compared tothe administration of melphalan or the control human IgG1.

[0120] Mode for Carrying Out the Invention

[0121] 1. Construction of a chimeric antibody

[0122] (1) Cloning of DNA encoding the V region of a mouse anti-HM 1.24monoclonal antibody

[0123] Preparation of mRNA

[0124] In order to clone DNA encoding the V region of a mouse anti-HM1.24 monoclonal antibody, the total RNA is prepared from a recoveredhybridoma using a known method such as a guanidine-ultracentrifugemethod (Chirgwin, J. M. et al., Biochemistry (1979), 18, 5294-5299), theAGPC method (Chomczynski, P. et al. (1987), 162, 156-159), etc. and mRNAis prepared using the Oligo(dT)-cellulose spun column etc. attached withthe mRNA Purification Kit (manufactured by Pharmacia), etc. Furthermore,by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia)mRNA can be prepared without the extraction step of the total RNA.

[0125] Preparation and Amplification of cDNA

[0126] From the mRNA obtained in the above-mentioned Preparation ofmRNA, each cDNA for the V regions of an L chain and an H chain issynthesized using a reverse transcriptase. The cDNA of the V region ofthe L chain is synthesized using the AMV Reverse TranscriptaseFirst-Strand cDNA Synthesis Kit. For the amplification of thesynthesized cDNA, an appropriate primer that hybridizes with the leadersequence and the C region of the antibody gene (for example, the MKVprimer having the base sequences represented by the SEQ ID NO: 29 to 39,and the MKC primer having the base sequence represented by the SEQ IDNO: 40).

[0127] The synthesis and amplification of the CDNA of the V region of anH chain can be carried out by PCR (polymerase chain reaction) by the5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA, 85,8998-9002, 1988, Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932,1989) using the 5′-Ampli FINDER RACE kit (CLONTECH). To the 5′-end ofthe cDNA synthesized as above, the Ampli FINDER Anchor is ligated, andas a primer for amplification of the V region of the H chain, a primerthat specifically hybridizes with the Anchor primer (SEQ ID NO: 77) andthe constant region (Cγ region) of a mouse H chain (for example, theMHC2a primer having the base sequence represented by SEQ ID NO: 42) canbe used.

[0128] Purification of DNA and the Determination of the Base SequenceThereof

[0129] An agarose gel electrophoresis is conducted on the PCR productusing a known method to excise the desired DNA fragment, and DNA isrecovered and purified therefrom, which is then ligated to a vector DNA.

[0130] DNA can be purified using a commercial kit (for example,GENECLEAN II; BIO101). A known vector DNA (for example, pUC19,Bluescript, etc.) can be used to retain DNA fragments.

[0131] The above DNA and the above DNA vector are ligated using a knownligation kit (manufactured by Takara Shuzo) to obtain a recombinantvector. The obtained recombinant vector is then introduced intoEscherichia coli JM109, after which ampicillin resistant colonies areselected and a vector DNA is prepared based on a known method (J.Sambrook, et al., “Molecular Cloning”, Cold Spring Harbor LaboratoryPress, 1989). After digesting the above vector DNA with restrictionenzymes, the base sequence of the desired DNA is determined by a knownmethod (for example, the dideoxy method) (J. Sambrook, et al.,“Molecular Cloning”, Cold Spring Harbor Laboratory Press, 1989). Inaccordance with the present invention, an automatic sequencing system(DNA Sequencer 373A; manufactured by ABI Co. Ltd.) can be used.

[0132] Complementarity Determining Region

[0133] The V region of an H chain and the V region of an L chain form anantigen binding site, of which overall structures have similarproperties. Thus, each of four framework regions (FR) has been ligatedby three hypervariable regions, i.e. complementarity determining regions(CDRs). The amino acid sequences of FRs have been relatively wellconserved whereas variation is extremely high among the amino acidsequences of CDR regions (Kabat, E. A. et al., “Sequence of Proteins ofImmunological Interest”, US Dept. Health and Human Services, 1983).

[0134] Many portions of the above four FRs take the β-sheet structurewith a result that three CDRs form loops. CDRs may sometimes form partof the β-sheet structure. The three CDRs are retained sterically inclose proximity with one another and form an antigen binding site withthree CDRs of the pairing region.

[0135] Based on these facts, the amino acid sequence of the variableregion of a mouse anti-HM 1.24 antibody is fitted to the data base ofthe amino acid sequences of antibodies prepared by Kabat et al.(“Sequence of Proteins of Immunological Interest”, US Dept. Health andHuman Services, 1983) to investigate homology and thereby to find CDRregions.

[0136] (2) Construction of Expression Vectors for a Chimeric Antibody

[0137] Once a DNA fragment encoding the V regions of the mouse L chainand H chain of a mouse monoclonal antibody is cloned, a chimeric anti-HM1.24 antibody can be obtained by linking these mouse V regions to a DNAencoding the constant region of a human antibody and then by expressingthem.

[0138] A basic method for constructing a chimeric antibody compriseslinking the mouse leader sequence and the V region sequence present inthe cloned cDNA to a sequence encoding the C region of a human antibodyalready present in an expression vector for mammalian cells.Alternatively it comprises linking the mouse leader sequence and the Vregion sequence present in the cloned cDNA to a sequence encoding the Cregion of a human antibody, which is then linked to an expression vectorfor mammalian cells.

[0139] The C region of a human antibody can be the C region of any Hchain and the C region of any L chain. There can be mentioned, forexample, Cγ1, Cγ2, Cγ3, or Cγ4 of a human H chain, or Cλ or Ck of an Lchain.

[0140] For production of a chimeric antibody two kinds of expressionvectors are constructed: they are an expression vector comprising DNAencoding the V region of a mouse L chain and the C region of a human Lchain under the control of an expression regulatory region such as theenhancer/promoter system, and an expression vector comprising DNAencoding the V region of a mouse H chain and the C region of a human Hchain under the control of an expression regulatory region such as theenhancer/promoter system. Subsequently, using these expression vectors ahost cell such as a mammalian cell is cotransformed, and the transformedcells are cultured in vitro or in vivo to produce a chimeric antibody(for example, WO 91-16928).

[0141] Alternatively, DNA encoding the mouse leader sequence and the Vregion of an L chain and the C region of a human L chain and DNAencoding the mouse leader sequence and the V region of an H chain andthe C region of a human H chain present in the cloned cDNA areintroduced into a single expression vector (see, InternationalApplication Publication No. WO 94-11523), and a host cell is transformedusing said vector. The transformed host is then cultured in vitro or invivo to produce the desired chimeric antibody.

[0142] 1) Construction of a Chimeric H Chain

[0143] An expression vector for the H chain of the chimeric antibody canbe obtained by introducing cDNA encoding the V region of a mouse H chaininto an appropriate expression vector containing genomic DNA or cDNAencoding the C region of the H chain of a human antibody. As the Cregion of an H chain there can be mentioned, for example, Cγ1, Cγ2, Cγ3,or Cγ4.

[0144] Construction of an Expression Vector for a Chimeric H ChainContaining Cγ1 Genomic DNA

[0145] As an expression vector having genomic DNA for Cγ1 as the Cregion of an H chain, there can be used, for example HFE-PMh-gγ1(International Application Publication No. WO 92/19759) orDHFR-ΔE-RVh-PM1f (International Application Publication No. WO92/19759).

[0146] In order to insert cDNA encoding the V region of a mouse H chaininto these expression vectors, suitable base sequences may be introducedusing the PCR method. These suitable base sequences can be introduced bythe PCR method using a PCR primer designed to have a recognitionsequence for a suitable restriction enzyme at the 5′-end and a Kozakconsensus sequence immediately before the start codon, and a PCR primerdesigned to have at the 3′-end a recognition sequence for a suitablerestriction enzyme and a splice donor site where a primary transcript ofgenomic DNA is properly spliced to become an mRNA.

[0147] The cDNA thus constructed encoding the V region of a mouse Hchain is treated with suitable restriction enzymes, inserted into theabove-mentioned expression vector, and a chimeric H chain-expressionvector comprising the Cγ1 DNA can be constructed.

[0148] Construction of an Expression Vector for the cDNA Chimeric HChain

[0149] An expression vector having the cDNA of Cγ1 as the C region of anH chain may be constructed as follows. Thus, it can be constructed bypreparing mRNA from a CHO cell in which the expression vectorDHFR-ΔE-RVh-PM1f (International Application Publication No. WO 92/19759)encoding genomic DNA of the V region of the H chain of a humanized PM1antibody and the C region Cγ1 of the H chain of a human antibody (N.Takahashi, et al., Cell 29, 671-679, 1982) and the expression vectorRV1-PM1a (International Application Publication No. WO 92/19759)encoding genomic DNA of the V region of the L chain of a humanized PM1antibody and the C region of the k chain of a human antibody L chainhave been integrated; cloning cDNA comprising the V region of the Hchain of the humanized PM1 antibody and the C region Cγ1 of the H chainof the human antibody by the RT-PCR method, and; ligating to a suitableexpression vector for animal cells using suitable restriction enzymesites.

[0150] In order to directly ligate cDNA encoding the V region of a mouseH chain to cDNA containing the C region Cγ1 of the H chain of a humanantibody, suitable base sequences can be introduced by the PCR method.For example, these suitable base sequences can be introduced by the PCRmethod using a PCR primer designed to have a recognition sequence for asuitable restriction enzyme at the 5′-end and a Kozak consensus sequenceimmediately before the start codon, and a PCR primer designed to have arecognition sequence for a suitable restriction enzyme used for 'directligation of the C region Cγ1 of an H chain at the 3′-end.

[0151] An expression vector containing a cDNA chimeric H chain can beconstructed by treating the cDNA thus constructed encoding the V regionof a mouse H chain with a suitable restriction enzyme, ligating to theabove-mentioned cDNA containing the C region Cγ1 of the H chain, andinserting to an expression vector such as pCOS1 or pCHO1.

[0152] 2) Construction of the L Chain of a Chimeric Antibody

[0153] An expression vector for the L chain of a chimeric antibody maybe obtained by linking cDNA encoding the V region of a mouse L chain togenomic DNA or cDNA encoding the C region of the L chain of a humanantibody, and then introducing it into a suitable expression vector. Asthe C region of an L chain there can be mentioned, for example a k chainor a λ chain.

[0154] Construction of an Expression Vector for the k Chain of a cDNAChimeric L Chain In order to construct an expression vector containingcDNA encoding the V region of a mouse L chain, suitable base sequencescan be introduced using the PCR method. For example, these suitable basesequences can be introduced by the PCR method using a PCR primerdesigned to have a recognition sequence for a suitable restrictionenzyme and a Kozak consensus sequence at the 5′-end, and a PCR primerdesigned to have a recognition sequence for a suitable restrictionenzyme at the 3′-end.

[0155] The K chain C region of a human L chain for linking to the Vregion of a mouse L chain can be constructed from, for exampleHEF-PM1k-gk (see International Application Publication No. WO 92/19759)containing genomic DNA. An expression vector for the K chain of the Lchain of a cDNA chimeric antibody can be constructed by introducingrecognition sequences of suitable restriction enzymes at the 5′-end or3′-end of DNA encoding the K chain C region of L chain by the PCRmethod, ligating the thus constructed V region of the mouse L chain tothe K chain C region of L chain, and then inserting into an expressionvector such as pCOS1 or pCHO1.

[0156] 2. Construction of a Reshaped Human Antibody

[0157] (1) Designing of the V region of a Reshaped Human anti-HM 1.24Antibody

[0158] In order to construct a reshaped human antibody in which the CDRof a mouse monoclonal antibody has been grafted to a human antibody, itis desirable that there is a high homology between the FR of the mousemonoclonal antibody and the FR of the human antibody. Thus, the Vregions of the L chain and the H chain of the mouse anti-HM 1.24antibody are compared to the V regions of all known antibodies of whichstructures have been elucidated, using the Protein Data Bank.

[0159] The V region of the L chain of a mouse anti-HM 1.24 antibody ismost similar to the consensus sequence of the subgroup IV of the Vregion of the L chain of a human antibody (HSGIV) with a homology of66.4%. On the other hand, it shows a homology of 56.9%, 55.8%, and 61.5%with HSGI, HSGII, and HSGIII, respectively.

[0160] The V region of L chain of a mouse anti-HM 1.24 antibody, whencompared to the V region of the L chain of known human antibodies, showsa homology of 67.0% with the V region of the L chain of the humanantibody REI, one of subgroup I of the V region of the L chain of thehuman antibody. Therefore, the FR of the REI was used as the startingmaterial for construction of the V region of the L chain of the reshapedhuman anti-HM 1.24 antibody.

[0161] Version a of the V region of the L chain of the reshaped humananti-HM 1.24 antibody was designed. In this version, the FR of the humanantibody was made identical with the REI-based FR present in thereshaped human CAMPATH-1H antibody (see Riechmann, L. et al., Nature322, 21-25, (1988), the FR contained in version a of the V region of theL chain of a reshaped human PM-1 antibody described in InternationalApplication Publication No. WO 92-19759), and the mouse CDR was madeidentical with the CDR in the V region of the L chain of the mouseanti-HM 1.24 antibody.

[0162] The V region of the H chain of a mouse anti-HM 1.24 antibody ismost similar to the consensus sequence of the V region of the H chain ofa human antibody (HSGI) with a homology of 54.7%. On the other hand, itshows a homology of 34.6% and 48.1% with HSGII and HSGIII, respectively.When the V region of the H chain of a mouse anti-HM 1.24 antibody iscompared to the V region of the H chain of known human antibodies, FR1to FR3 were most similar to the V region of the H chain of the humanantibody HG3, one of subgroup I of the V region of a human H chain(Rechavi, G. et al., Proc. Natl. Acad. Sci. USA, 80, 855-859), with ahomology of 67.3%.

[0163] Therefore, the FR of the human antibody HG3 was used as thestarting material for construction of the V region of the H chain of areshaped human anti-HM 1.24 antibody.

[0164] However, since the amino acid sequence of the FR4 of the humanantibody HG3 has not been described, the amino acid sequence of the FR4of the human antibody JH6 (Ravetch, J.V. et al., Cell, 27, 583-591) thatshows the highest homology with the FR4 of a mouse anti-HM 1.24 antibodywas used as FR4. The FR4 of JR6 has the same amino acid sequence as theFR4 of the H chain of a mouse anti-HM 1.24 antibody except only oneamino acid.

[0165] In the first version a of the V region of the H chain of areshaped human anti-HM 1.24 antibody, FRi to FR3 were made identicalwith the FRI to FR3 of the human antibody HG3, except that the aminoacids at position 30 in the human FRI and position 71 in the human FR3were made identical with the amino acids of the mouse anti-HM 1.24antibody, and the CDR was made identical with the CDR in the V region ofthe H chain of a mouse anti-HM 1.24 antibody.

[0166] (2) Construction of the V region of the L Chain of a ReshapedHuman anti-HM 1.24 Antibody

[0167] The L chain of a reshaped human anti-HM 1.24 antibody isconstructed by the CDR grafting in the PCR method. The method isschematically shown in FIG. 4. Eight PCR primers are used forconstruction of a reshaped human anti-HM 1.24 antibody (version a)having the FR derived from the human antibody REI. The external primersA (SEQ ID NO: 47) and H (SEQ ID NO: 48) are designed to hybridize withthe DNA sequence of the HEF expression vector HEF-VL-gk .

[0168] The CDR grafting primers L1S (SEQ ID NO: 49), L2S (SEQ ID NO:50), and L3S (SEQ ID NO: 51) have a sense DNA sequence. The CDR graftingprimers L1A (SEQ ID NO: 52), L2A (SEQ ID NO: 53), and L3A (SEQ ID NO:54) have an antisense DNA sequence, each having a complementary DNAsequence (20 to 23 bp) to the DNA sequence at the 5′-end of the primersL1S, L2S, and L3S, respectively.

[0169] In the first stage of PCR, the four reactions A-L1A, L1S-L2A,L2S-L3A, and L3S-H are conducted and each PCR product purities. The fourPCR products from the first PCR are allowed to assemble with one anotherby their own complementarity (see WO 92-19759). Then, the externalprimers A and H are added to amplify the full-length DNA encoding the Vregion of the L chain of a reshaped human anti-HM 1.24 antibody (thesecond PCR). In the above-mentioned PCR, the plasmid HEF-RVL-M21a (seeInternational Application Publication No. WO 95-14041) encoding theversion a of the V region of the L chain of a reshaped human ONS-M21antibody based on the human antibody REI-derived FR can be employed as atemplate.

[0170] In the first stage of PCR, template DNA and each of primers wereused.

[0171] PCR products A-L1A (215 bp), L1S-L2A(98 bp), L2S-L3A (140 bp),and L3S-H (151 bp) are purified using 1.5% low melting point agarose geland are assembled in the second PCR. In the second PCR, each productfrom the first PCR and each external primer (A and H) are used.

[0172] A 516 bp DNA fragment resulting from the second PCR is purifiedusing 1.5% low melting point agarose gel, digested with BamHI andHindIII, and the DNA fragments thus obtained are cloned into the HEFexpression vector HEF-VL-gK. After determining the DNA sequence, theplasmid containing the DNA fragment having the correct amino acidsequence of the V region of the L chain of a reshaped human anti-HM 1.24antibody was termed the plasmid HEF-RVLa-AHM-gk . The amino acidsequence and the base sequence of the V region of the L chain containedin this plasmid HEF-RVLa-AHM-gk are shown in SEQ ID NO: 9.

[0173] The version b of the V region of the L chain of a reshaped humananti-HM 1.24 antibody can be constructed by mutagenesis using PCR.Mutagen primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) are sodesigned as to mutate phenylalanine at position 71 to tyrosine.

[0174] After the above primers are amplified using the plasmidHEF-RVLa-AHM-gk as a template, the final product is purified. Bydigesting with BamHI and HindIII, the DNA fragments obtained are clonedinto the HEF expression vector HEF-VL-gK to obtain plasmidHEF-RVLb-AHM-gk. The amino acid sequence and the base sequence of the Vregion of the L chain contained in this plasmid HEF-RVLb-AHM-gk areshown in SEQ ID NO: 10.

[0175] (3) Construction of the V region of the H Chain of a ReshapedHuman Anti-HM 1.24 Antibody

[0176] 3-1. Construction of Versions a to e of the V Region of the HChain of a Reshaped Human anti-HM 1.24 antibody

[0177] DNA encoding the V region of the H chain of a reshaped humananti-HM 1.24 antibody can be designed as follows. By linking the DNAsequence encoding the FRs 1 to 3 of the human antibody HG3 and the FR4of the human antibody JH6 to the DNA sequence encoding the CDR of the Vregion of the H chain of a mouse anti-HM 1.24 antibody, a full lengthDNA encoding the V region of the H chain of a reshaped human anti-HM1.24 antibody may be designed.

[0178] Then, the HindIII recognition site/KOZAK consensus sequence andthe BamHI recognition site/splice donor sequence, respectively, areattached to the 5′-end and the 3′-end of this DNA sequence so as toallow insertion of the HEF expression vector.

[0179] The DNA sequence thus designed is divided into fouroligonucleotides. Subsequently, oligonucleotides which potentiallyhinder the assembly of these oligonucleotides are subjected to computeranalysis for the secondary structure.

[0180] The sequences of the four oligonucleotides RVH1 to RVH4 are setforth in SEQ ID NO: 57 to 60. These oligonucleotides have a length of119 to 144 bases and have a 25 to 26 bp overlapping region. Among theoligonucleotides, RVH2 (SEQ ID NO: 58) and RVH4 (SEQ ID NO: 60) have asense DNA sequence, and RVH1 (SEQ ID NO: 57) and RVH3 (SEQ ID NO: 59)have an antisense DNA sequence. The method for assembling these fouroligonucleotides by the PCR method is shown in the figure (see FIG. 5).

[0181] PCR is carried out using the four oligonucleotides and RHP1 (SEQID NO: 60) and RHP2 (SEQ ID NO: 62) as the external primers.

[0182] The amplified 438 bp DNA fragment is purified, digested withHindIII and BamHI, and then cloned into the HEF expression vectorHEF-VH-gγ1. After determination of the base sequence, the plasmid thatcontains the DNA fragment encoding the correct amino acid sequence ofthe V region of the H chain was termed HEF-RVHa-AHM-gγ1. The amino acidsequence and the base sequence of the V region of the H chain containedin this plasmid HEF-RVHa-AHM-gγ1 are shown in SEQ ID NO: 11.

[0183] Each of versions b, c, d, and e of the V region of the H chain ofa reshaped human anti-HM 1.24 antibody is constructed as follows. Inconstructing each of version b and after of the V region of the H chainof a reshaped human anti-HM 1.24 antibody, a three-dimensionalstructural model of the V region of a mouse anti-HM 1.24 antibody can beconstructed in order to predict the position of the amino acid residueto be substituted in the antibody molecule.

[0184] Using as the mutagen primer BS (sequence 63) and BA (SEQ ID NO:64) designed to mutate arginine at position 66 to lysine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version bis amplified to obtain plasmid HEF-RVHb-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHb-AHM-gγ1 are shown in SEQ ID NO: 12.

[0185] Using as the mutagen primer CS (sequence 65) and CA (SEQ ID NO:66) designed to mutate threonine at position 73 to lysine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version cis amplified to obtain plasmid HEF-RVHc-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHc-AHM-gγ1 are shown in SEQ ID NO: 13.

[0186] Using as the mutagen primer DS (sequence 67) and DA (SEQ ID NO:68) designed to mutate arginine at position 66 to lysine and threonineat position 73 to lysine and as a template DNA the plasmidHEF-RVHa-AHM-gγ1 by the PCR method, version d is amplified to obtainplasmid HEF-RVHd-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHd-AHM-gγ1 are shown in SEQ ID NO: 14.

[0187] Using as the mutagen primer ES (sequence 69) and EA (SEQ ID NO:70) designed to mutate valine at position 67 to alanine and methionineat position 69 to leucine and as a template DNA the plasmidHEF-RVHa-AHM-gγ1, version e is amplified to obtain plasmidHEF-RVHe-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHe-AHM-gγ1 areshown in SEQ ID NO: 15.

[0188]3-2. Construction of the H Chain Hybrid V Region

[0189] By constructing a H chain hybrid V region, it is possible toinvestigate which FR of the V region of a humanized antibody contributesto the binding activity and the binding inhibition activity. Among thetwo that were constructed, the amino acid sequences of FR1 and FR2 arederived from a mouse anti-HM 1.24 antibody and those of FR3 and FR4 arefrom version a of the V region of the H chain of a reshaped humananti-HM 1.24 antibody (mouse human hybrid anti-HM 1.24 antibody) in one,and the amino acid sequences of FR1 and FR2 are derived from version aof the V region of the H chain of a reshaped human anti-HM 1.24 antibodyand those of FR3 and FR4 are from a mouse anti-HM 1.24 antibody (humanmouse hybrid anti-HM 1.24 antibody) in the other. The amino acidsequences of the CDR regions are all derived from a mouse anti-HM 1.24antibody.

[0190] Two H chain hybrid V regions are constructed by the PCR method.The method is schematically shown in FIG. 6 and 7. For the constructionof two H chain hybrid V regions four primers can be used. The externalprimers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) are designed tohybridize with the DNA sequence of the HEF expression vector HEF-VH-gγ1.The H chain hybrid construction primer HYS (SEQ ID NO: 73) is designedto have the sense DNA sequence and the H chain hybrid primer HYA (SEQ IDNO: 74) to have the antisense DNA sequence so that the DNA sequences arecomplementary to each other.

[0191] For the construction of the H chain hybrid V region in which theamino acid sequences of FR1 and FR2 are derived from a mouse anti-HM1.24 antibody and those of FR3 and FR4 are from version a of the Vregion of the H chain of a reshaped human anti-HM 1.24 antibody, PCRusing the plasmid HEF-1.24H-gγ1 as a template, the external primer a,and the H chain hybrid primer HYA, and PCR using the plasmidHEF-RVHa-AHM-gγ1 as a template, the H chain hybrid primer HYA, and theexternal primer h are carried out in the first stage of PCR and each PCRproduct is purified.

[0192] The two PCR products from the first PCR are allowed to assembleby their own complementarity (see International Application PublicationNo. WO 92-19759). Then, by adding the external primers a and h, afull-length DNA encoding the H chain hybrid V region in which the aminoacid sequences of FR1 and FR2 are derived from a mouse anti-HM 1.24antibody and those of FR3 and FR4 are from version a of the V region ofthe H chain of a reshaped human anti-HM 1.24 antibody is amplified inthe second PCR stage.

[0193] For the construction of the H chain hybrid V region in which theamino acid sequences of FR1 and FR2 are derived from version a of the Vregion of the H chain of a reshaped human anti-HM 1.24 antibody andthose of FR3 and FR4 are from a mouse anti-HM 1.24 antibody, PCR usingthe plasmid HEF-RVHa-AHM-gγ1 as a template, the external primer a, andthe H chain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-gγ1as a template, the H chain hybrid primer HYS, and the external primer hare carried out in the first stage of PCR and each PCR product ispurified.

[0194] The two PCR purified products from the first PCR are allowed toassemble by their own complementarity (see International ApplicationPublication No. WO 92-19759). Then, by adding the external primers a andh, a full-length DNA encoding the H chain hybrid V region in which theamino acid sequences of FR1 and FR2 are derived from version a of the Vregion of the H chain of a reshaped human anti-HM 1.24 antibody andthose of FR3 and FR4 are from a mouse anti-HM 1.24 antibody is amplifiedin the second PCR stage.

[0195] The methods of the first PCR, purification of PCR products,assembling, the second PCR, and cloning into the HEF expression vectorHEF-VH-gγ1 are carried out according to the method shown in “Example 9.Construction of the V region of the L chain of a reshaped human anti-HM1.24 antibody”. After determination of the DNA sequence, the plasmidthat contains the DNA fragment encoding the correct amino acid sequenceof the H chain hybrid V region in which the amino acid sequences of FR1and FR2 are derived from a mouse anti-HM 1.24 antibody and those of FR3and FR4 are from version a of the V region of the H chain of a reshapedhuman anti-HM 1.24 antibody was termed HEF-MH-RVH-AHM-gγ1.

[0196] The amino acid sequence and the base sequence of the V region ofthe H chain contained in this plasmid HEF-MH-RVH-AHM-gγ1 are shown inSEQ ID NO: 75. Also, the plasmid that contains the DNA fragment encodingthe correct amino acid sequence of the H chain hybrid V region in whichthe amino acid sequences of FR1 and FR2 are derived from a version areshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are fromthe V region of-the H chain of a mouse anti-HM 1.24 antibody was termedHEF-HM-RVH-AHM-gγ1. The amino acid sequence and the base sequence of theV region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gγ1 areshown in SEQ ID NO: 76.

[0197] 3-3. Construction of Versions f to s of the V Region of the HChain of a Reshaped Human anti-HM 1.24 antibody

[0198] Each of versions f, g, h, i, j, k, l, m, n, o, p, q, r, and s ofthe V region of the H chain of a reshaped human anti-HM 1.24 antibody isconstructed as follows. In constructing each of versions f and after ofthe V region of the H chain of a reshaped human anti-HM 1.24 antibody, athree-dimensional structural model of the V region of a mouse anti-HM1.24 antibody can be constructed, as mentioned above, in order topredict the position of the amino acid residue to be substituted in theantibody molecule.

[0199] Using as the mutagen primer FS (sequence 78) and FA (SEQ ID NO:79) designed to mutate threonine at position 75 to serine and valine atposition 78 to alanine and as a template DNA the plasmidHEF-RVHe-AHM-gγ1 by the PCR method, version f is amplified to obtainplasmid HEF-RVHf-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHf-AHM-gγ1 are shown in SEQ ID NO: 16.

[0200] Using as the mutagen primer GS (sequence 80) and GA (SEQ ID NO:81) designed to mutate alanine at position 40 to arginine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version g is amplified toobtain plasmid HEF-RVHg-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHg-AHM-gγ1 are shown in SEQ ID NO: 17.

[0201] Using as the mutagen primer FS and FA and as a template DNA theplasmid HEF-RVHb-AHM-gγ1, version h is amplified to obtain the plasmidHEF-RVHh-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHh-AHM-gγ1 areshown in SEQ ID NO: 18.

[0202] Using as the mutagen primer IS (sequence 82) and IA (SEQ ID NO:83) designed to mutate arginine at position 83 to alanine and serine atposition 84 to phenylalanine and as a template DNA the plasmidHEF-RVHh-AHM-gγ1, version i is amplified to obtain plasmidHEF-RVHi-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHi-AHM-gγ1 areshown in SEQ ID NO: 19.

[0203] Using as the mutagen primer JS (SEQ ID NO: 84) and JA (SEQ ID NO:85) designed to mutate arginine at position 66 to lysine and as atemplate DNA the plasmid HEF-RVHf-AHM-gγ1, version j is amplified toobtain plasmid HEF-RVHj-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHj-AHM-gγ1 are shown in SEQ ID NO: 20.

[0204] Using as the mutagen primer KS (SEQ ID NO: 86) and KA (SEQ ID NO:87) designed to mutate glutamic acid at position 81 to glutamine and asa template DNA the plasmid HEF-RVHh-AHM-gγ1, version k is amplified toobtain plasmid HEF-RVHk-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHk-AHM-gγ1 are shown in SEQ ID NO: 21.

[0205] Using as the mutagen primer LS (SEQ ID NO: 88) and LA (SEQ ID NO:89) designed to mutate glutamic acid at position 81 to glutamine andserine at position 82B to isoleucine and as a template DNA the plasmidHEF-RVHh-AHM-gγ1, version 1 is amplified to obtain plasmidHEF-RVHl-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHl-AHM-gγ1 areshown in SEQ ID NO: 22.

[0206] Using as the mutagen primer MS (SEQ ID NO: 90) and MA (SEQ ID NO:91) designed to mutate glutamic acid at position 81 to glutamine, serineat position 82b to isoleucine, and threonine at position 87 to serineand as a template DNA the plasmid HEF-RVHh-AHM-gγ1, version m isamplified to obtain plasmid HEF-RVHm-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHm-AHM-gγ1 are shown in SEQ ID NO: 23.

[0207] Using as the mutagen primer NS (SEQ ID NO: 92) and NA (SEQ ID NO:93) designed to mutate serine at position 82B to isoleucine and as atemplate DNA the plasmid HEF-RVHh-AHM-gγ1, version n is amplified toobtain plasmid HEF-RVHn-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHn-AHM-gγ1 are shown in SEQ ID NO: 24.

[0208] Using as the mutagen primer OS (SEQ ID NO: 94) and OA (SEQ ID NO:95) designed to mutate threonine at position 87 to serine and as atemplate DNA the plasmid HEF-RVHh-AHM-gγ1, version o is amplified toobtain plasmid HEF-RVHo-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHo-AHM-gγ1 are shown in SEQ ID NO: 25.

[0209] Using as the mutagen primer PS (SEQ ID NO: 96) and PA (SEQ ID NO:97) designed to mutate valine at position 78 to alanine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version p is amplified by thePCR method to obtain plasmid HEF-RVHp-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHp-AHM-gγ1 are shown in SEQ ID NO: 26.

[0210] Using as the mutagen primer QS (SEQ ID NO: 98) and QA (SEQ ID NO:99) designed to mutate threonine at position 75 to serine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version q is amplified by thePCR method to obtain plasmid HEF-RVHq-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHq-AHM-gγ1 are shown in SEQ ID NO: 27.

[0211] Using as the mutagen primer CS (SEQ ID NO: 65) and CA (SEQ ID NO:66) and as a template DNA the plasmid HEF-RVHp-AHM-gγ1, version r isamplified by the PCR method to obtain plasmid HEF-RVHr-AHM-gγ1. Theamino acid sequence and the base sequence of the V region of the H chaincontained in this plasmid HEF-RVHr-AHM-gγ1 are shown in SEQ ID NO: 28.

[0212] Using as the mutagen primer SS (SEQ ID NO: 100) and SA (SEQ IDNO: 101) designed to mutate methionine at position 69 to isoleucine andas a template DNA the plasmid HEF-RVHr-AHM-gγ1, version s is amplifiedto obtain plasmid HEF-RVHs-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHs-AHM-gγ1 are shown in SEQ ID NO: 102.

[0213] The amino acid sequences of the V region of the L chainconstructed are shown in Table 1, and the amino acid sequences of the Vregion of the H chain are shown in Tables 2 to 4. TABLE 1 The amino acidsequence of the V region of the L chain  FR1                     CDR1        FR2         1         2          3          4 1234567890123456789012345678901234 567890123456789 AHM DIVMTQSHKFMSTSVGDRVSITC KASQDVNTAVAWYQQKPGQSPKLLIY HuSG IDIQMTQSPSSLSASVGDRVTITC             WYQQKPGKAPKLLIY REIDIQMTQSPSSLSASVGDRVTITC             WYQQKPGKAPKLLIY RVLa----------------------- ----------- --------------- RVLb----------------------- ----------- ---------------  CDR2     FR35          6         7         8 012345678901234567890123456789012345678 AHM SASNRYTGVPDRITGSGSGTDFTFTISSVQAEDLALYYC HuSG I        GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC REI        GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC RVLa --------------------------------------- RVLb ---------------------Y-----------------   CDR3      FR4  9         10 9012345678901234567 AHM QQHYSTPFT FGSGTKLEIK HuSG I           FGQGTKVEIK REI          FGQGTKVEIK RYLa --------- ---------- RVLb --------- ----------

[0214] TABLE 2 The amino acid sequence of the V region of the H chain(1)   FR1                           CDR1   FR2         1         2         3           4123456789012345678901234567890 12345 67890123456789 AHMQVQLQQSGAELARPGASVKLSCKASGYTFT PYWMQ WVKQRPGQGLEWIG HuSGIEVQLVQSGADVKKPGXSVXVSCKASGYTFS       WVRQAPGXGLDWVG HG3QVQLVQSGAEVKKPGASVKVSCKASGYTFN       WVRQAPGQGLEWMG RVHa-----------------------------T ----- -------------- RVHb-----------------------------T ----- -------------- RVHc-----------------------------T ----- -------------- RVHd-----------------------------T ----- -------------- RVHe-----------------------------T ----- -------------- RVHf-----------------------------T ----- -------------- RVHg-----------------------------T ----- ----R--------- RVHh-----------------------------T ----- -------------- RVHi-----------------------------T ----- -------------- RVHj-----------------------------T ----- -------------- RVHk-----------------------------T ----- -------------- RVHl-----------------------------T ----- -------------- RVHm-----------------------------T ----- -------------- RVHn-----------------------------T ----- -------------- RVHo-----------------------------T ----- -------------- RVHp-----------------------------T ----- -------------- RVHq-----------------------------T ----- -------------- RVHr-----------------------------T ----- -------------- RVHs-----------------------------T ----- --------------

[0215] TABLE 3 The amino acid sequence of the V region of the H chain(2)   CDR2              FR35          6          7         8            9 012A345678901234567890123456789012ABC345678901234 AHM SIFPGDGDTRYSQKFKGKATLTADKSSSTAYMQLSILAFEDSAVYYCAR HuSG I                  RVTXTXDXSXNTAYMELSSLRSEDTAVYYCAR HG3                  RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR RVHa----------------- -----A-------------------------- RVHb----------------- K----A-------------------------- RVHc----------------- -----A-K------------------------ RVHd----------------- K----A-K------------------------ RVHe----------------- -A-L-A-------------------------- RVHf----------------- -A-L-A---S--A------------------- RVHg----------------- -----A-------------------------- RVHh----------------- K----A---S--A------------------- RVHi----------------- K----A---S--A-------AF---------- RVHj----------------- KA-L-A---S--A------------------- RVHk----------------- K----A---S--A--Q---------------- RYHl----------------- K----A---S--A--Q--I------------- RVHm----------------- K----A---S--A--Q--I-----S------- RVHn----------------- K----A---S--A-----I------------- RVHo----------------- K----A---S--A-----------S------- RVHp----------------- -----A------A------------------- RVHq----------------- -----A---S---------------------- RVHr----------------- -----A-K----A------------------- RVHs----------------- ---I-A-K----A-------------------

[0216] TABLE 4 The amino acid sequence of the V region of the H chain(3)  CDR3        FR4    10             11 57890ABJK12  34567890123 AHMGLRRGGYYFDY  WGQGTTLTVSS HuSGI              WGQGTLVTVSS JH6             WGQGTTVTVSS RVHa ----------- ------------ RVHb ----------------------- RVHc ----------- ------------ RVHd ----------- ------------RVHe ----------- ------------ RVHf ----------- ------------ RVHg----------- ------------ RVHh ----------- ------------ RVHi ----------------------- RVHj ----------- ------------ RVHk ----------- ------------RVHl ----------- ------------ RVHm ----------- ------------ RVHn----------- ------------ RVHo ----------- ------------ RVHp ----------------------- RVHq ----------- ------------ RVHr ----------- ------------RVHs ----------- ------------

[0217] 3. Production of a Chimeric Antibody and a Reshaped HumanAntibody

[0218] For the production of a chimeric antibody or a reshaped humanantibody, two expression vectors for each are constructed, whichcomprises an expression vector comprising DNA encoding the V region of amouse H chain and the C region of a human H chain under the control ofan expression regulatory region such as the enhancer/promoter system andDNA encoding the V region of a mouse L chain and the C region of a humanL chain under the control of an expression regulatory region such as theenhancer/promoter system, or an expression vector comprising DNAencoding the V region of a humanized H chain and the C region of a humanH chain under the control of an expression regulatory region such as theenhancer/promoter system and DNA encoding the V region of a humanized Lchain and the C region of a human L chain under the control of anexpression regulatory region such as the enhancer/promoter system.

[0219] Subsequently, a host cell such as the mammalian cell iscotransformed using these vectors, and the transformed cells arecultured in vitro or in vivo to produce a chimeric antibody or areshaped human antibody (for example, International ApplicationPublication No. WO 91-16928). Furthermore, an antibody gene isintroduced into mammals such as goat to produce a transgenic animal,from the milk of which a chimeric antibody or a reshaped human antibodycan be obtained.

[0220] Also, the V region of an H chain and the C region of an H chain ,and the V region of an L chain and the C region of an L chain areligated to a single vector to transform a suitable host cell and therebyto produce antibodies. Thus, for the expression of chimeric antibodies,DNA encoding the mouse leader sequence and the V region of the H chainand human H chain C region present in the cloned cDNA, and DNA encodingthe mouse leader sequence and L chain V region and human L chain Cregion are introduced into a single expression vector (see InternationalApplication Publication No. WO 94-11523).

[0221] For the expression of a reshaped human antibody, DNA encoding theV region of a humanized H chain and C region of a human H chain, and DNAencoding the V region of a humanized L chain and the C region of a humanL chain are introduced into a single expression vector (seeInternational Application Publication No. WO 94-11523). Using saidvector a host cells are transformed, and the transformed host cells arecultured in vivo or in vitro to produce the desired chimeric antibody orthe reshaped human antibody.

[0222] A transformant that was transformed, as mentioned above, by agene encoding the desired chimeric antibody or a reshaped human antibodyis cultured, and the chimeric antibody or the reshaped human antibodyproduced can be isolated from the inside or the outside of the cells andpurified to homogeneity.

[0223] The isolation and purification of the desired protein of thepresent invention, a chimeric antibody or a reshaped human antibody, maybe carried out using an affinity column. As a column that employsprotein A, for example, there is mentioned HyperD, POROS, Sepharose F.F, etc. Alternatively, the conventional isolation and purificationmethods used for proteins can be used and the method is not limited inany way. For example, combinations of various chromatographic methods,ultrafiltration, salting-out, dialysis, and the like, as appropriate,would permit the isolation and purification of the chimeric antibody ofthe reshaped human antibody.

[0224] For the production of the chimeric anti-HM 1.24. antibody or thereshaped human anti-HM 1.24 antibody of the present invention, anyexpression method can be used including, for example, the eukaryoticcells such as animal cells, an established mammalian cell-line system,an insect cell system, a fungal cell system, and a yeast cell system,and the procaryotic cells such as bacterial cells such as Escherichiacoli cells, and the like. Preferably, the chimeric antibody or thereshaped human antibody of the present invention may be expressed in theCOS cells, the CHO cells, the Hela cells, the Vero cells, the myelomacells or the BHK cells.

[0225] In these cases, common promoters that are useful for theexpression of mammalian cells can be used. For example, preferably thehuman cytomegalovirus immediate early (HCMV) promoter may be used.Examples of the expression vectors containing the HCMV promoter includethose which are HCMV-VH-HCγ1, HCMV-VL-HCk , etc. and which are derivedfrom pSV2neo (International Application Publication No. WO 92-19759).

[0226] Furthermore, as a promoter for gene expression in the mammaliancells for use in the present invention, there can be used viralpromoters such as retrovirus, polyoma virus, adenovirus, simian virus 40(SV40), etc., and promoters derived from mammalian cells such as humanpolypeptide chain elongation factor 1α (HEF-1α), etc. For example, whenthe promoter of SV40 is used, expression can be easily carried out usingthe method of Mulligan et al. (Nature 277, 108(1979)), and when HEF-1αpromoter is used the method of Mizushima, S. et al. (Nucleic AcidsResearch, 18, 5322, 1990) can be used.

[0227] As a source of replication, there can be used those derived fromSV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and thelike, and for the amplification of the copy number of the gene in a hostcell system, the expression vector can include, as a selective marker,aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK)gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene,dihydrofolate reductase (DHRF) gene, and the like.

[0228] 4. The binding Inhibition Activity of a Chimeric Antibody or aReshaped Human Antibody

[0229] (1) Measurement of Antibody concentration

[0230] The concentration of purified antibody may be measured by ELISAor the measurement of absorbance.

[0231] ELISA plates for measurement of antibody concentration may beprepared as follows. Each well of a 96-well ELISA plate (for exampleMaxisorp, manufactured by NUNC) is immobilized with 100 μl of goatanti-human IgG antibody at a concentration of 1 μg/ml.

[0232] After blocking with 100 μg/ml of a dilution buffer (for example50 mM Tris-HCl, 1 mM MgCl₂, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN₃, 1%bovine serum albumin (BSA), pH 8.1), serial dilutions of culturesupernatant of cells in which the chimeric antibody, the hybridantibody, or the reshaped human antibody was expressed, for example theculture supernatant of COS cells or CHO cels, or the purified chimericantibody, hybrid antibody, or reshaped human antibody is added to eachwell. Then 100 μl of alkaline phosphatase conjugated goat anti-human IgGantibody is added, 1 mg/ml of the substrate solution (Sigma104,p-nitrophenyl phosphate, manufactured by SIGMA) is added, and then theabsorbance at 405 nm is measured using a microplate reader (Bio Rad). Asthe standard for the measurement of concentration, a human IgG1k(manufactured by The BInding Site) can be used. The concentration of thepurified antibody is obtained by measuring absorbance at 280 nm andcalculating with 1 mg/ml as 1.35 OD.

[0233] (2) Binding Activity

[0234] Binding activity can be measured by the Cell-ELISA using thehuman amniotic cell line WISH (ATCC CCL25). The Cell-ELISA plate may beprepared as follows. WISH cells prepared at an appropriate concentrationwith PRMI 1640 medium supplemented with 10% fetal bovine serum are addedto a 96-well plate, incubated overnight, and after washing twice withPBS(-), are fixed with 0.1% glutaraldehyde (manufactured by Nakalaitesque).

[0235] After blocking, 100 μl of serial dilutions of the culturesupernatant of cells in which the chimeric anti-HM 1.24 antibody, thehybrid anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibodywas expressed, for example the culture supernatant of COS cells or CHOcells, or the purified chimeric anti-HM 1.24 antibody, hybrid anti-HM1.24 antibody or reshaped human anti-HM 1.24 antibody is added to eachwell, incubated at room temperature for two hours, and then peroxidase-labelled rabbit anti-human IgG antibody (manufactured by DAKO) isadded.

[0236] After icubating at room temperature for one hour, the substratesolution is added and then incubated. Subsequently, the reaction isstopped by 50 μl of 6N sulfuric acid, and then absorbance at 490 nm ismeasured using the MICROPLATE READER Model 3550 (manufactured byBio-Rad).

[0237] (3) Measurement of Binding Inhibition Activity

[0238] Binding inhibition activity by the biotinylated mouse anti-HM1.24 antibody is measured by the Cell-ELISA using the human amnioticcell line WISH (ATCC CCL25). The Cell-ELISA plate may be preparedaccording to the above-mentioned (2). WISH cells prepared at anappropriate concentration with PRMI 1640 medium supplemented with 10%fetal bovine serum are added to a 96-well plate, incubated overnight,and after washing twice with PBS(-), are fixed with 0.1% glutaraldehyde(manufactured by Nakalai tesque).

[0239] After blocking, 50 μl of serial dilutions of the culturesupernatant of cells in which the chimeric anti-HM 1.24 antibody, thehybrid anti-HM 1.24 antibody or the reshaped human anti-HM 1.24 antibodywas expressed, for example the culture supernatant of COS cells or CHOcells, or the purified chimeric anti-HM 1.24 antibody, hybridanti-HI1.24 antibody or reshaped human anti-HM 1.24 antibody is added toeach well, and simultaneously 50 μl of 2 μg/ml biotinylated mouseanti-HM 1.24 antibody is added, and then incubated at room temperaturefor two hours, and after washing, peroxidase-labelled streptavidin(manufactured by DAKO) is added.

[0240] After icubating at room temperature for one hour and afterwashing, the substrate solution is added and then incubated.Subsequently, the reaction is stopped by 50 μl of 6N sulfuric acid, andthen absorbance at 490 nm is measured using the MICROPLATE READER Model3550 (manufactured by Bio-Rad).

[0241] Measurement of ADCC Activity

[0242] The ADCC activity of the chimeric antibody or the reshaped humanantibody of the present invention can be measured as follows. First,mononuclear cells are separated from human peripheral blood or bonemarrow by the density centrifugation method and prepared as the effectorcell. Human myeloma cells are prepared as the target cell by labellingthe RPMI 8226 cells (ATCC CCL 155) with ⁵¹Cr. Then, the chimericantibody or the reshaped human antibody to be measured for ADCC activityis added to the labelled target cells and incubated, and then a suitableratio of the effector cell is added to the target cell and incubated.

[0243] After incubation the supernatant is taken to be measured forradioactivity using a gamma counter. At this time, 1% NP-40 can be usedfor measurement of the maximum released radioactivity. Cytotoxicity (%)can be calculated as (A−C)/(B−C)×100, wherein A is radioactivity (cpm)released in the presence of antibody, B is radioactivity (cpm) releasedby NP-40, and C is radioactivity (cpm) released by the culture liquidalone without antibody.

[0244] When ADCC activity or CDC activity is expected for the C regionof antibody, human Cγ1 or human Cy3can be used as the C region ofantibody. Furthermore, by adding, altering, or modifying part of theamino acid of the C region of antibody, a higher ADCC activity or CDCactivity can be induced.

[0245] For example, there are the IgM-like polymerization of IgG byamino acid substitution (Smith, R.I.F. & Morrison, S.L, BIO/TECHNOLOGY(1994) 12, 683-688), the IgM-like polymerization of IgG by amino acidaddition (Smith, R.I.F. et al., J. Immunol. (1995) 154, 2226-2236),expression by tandem linking of genes encoding an L chain (Shuford, W.et al., Science (1991) 252, 724-727), dimerization of IgG by amino acidsubstitution (Caron, P.C. et al., J. Exp. Med. (1992) 176, 1191-1195,Shopes, B.J. Immunology (1992) 148, 2918-2922, dimerization of IgG bychemical modification (Wolff, E.A. et al., Cancer Res. (1993) 53,2560-2565), and the introduction of the effector function by amino acidalteration at the hinge region of antibodies (Norderhaug, L. et al.,Eur. J. Immunol (1991) 21, 2379-2384). They can be accomplished by theoligomer site directed mutagenesis using primers, addition of basesequences using restriction enzyme cleavage sites, and chemicalmodifiers that induces covalent bonding. in vivo diagnostics for Myeloma

[0246] The chimeric anti-HM 1.24 antibody or the reshaped human anti-HM1.24 antibody of the present invention can be used as an in vivodiagnostics for myeloma by linking it to a labelled compound such asradioisotope and the like.

[0247] Furthermore, fragments of the chimeric anti-HM 1.24 antibody orthe reshaped human anti-HM 1.24 antibody, such as Fab, F(ab′)2, Fv, orsingle chain Fv (scFv) wherein the Fv or the Fv of an H chain and an Lchain are linked by a suitable linker that has been bound to a labelcompound such as radioisotope etc. can be used as an in vivo diagnosticsfor myeloma.

[0248] Specifically these antibody fragments can be obtained byconstructing the gene encoding these antibody fragments, introducingthem into an expression vector, and then expressing in a suitable hostcells, or digesting the chimeric anti-HM 1.24 antibody or the reshapedhuman anti-HM 1.24 antibody with a suitable enzyme.

[0249] The above-mentioned in vivo diagnostics for myeloma can besystematically administered in a parenteral manner.

[0250] A pharmaceutical composition and a therapeutic agent for myelomaIn order to confirm the therapeutic effects of the chimeric anti-HM 1.24antibody or the humanized anti-HM 1.24 antibody of the presentinvention, said antibodies are administered to a myelomacells-transplanted animal and the anti-tumor effects are evaluated.

[0251] As myeloma cells to be transplanted to animals, human myelomacells are preferred, and there can be mentioned, for example, KPMM2(Japanese Unexamined Patent Publication (Kokai) No. 7-236475), RPMI8226(ATCC CCL 155), ARH-77 (ATCC CRL 1621), and S6B45 (Suzuki, H. et al.,Eur. J. Immunol. (1992) 22, 1989-1993). As the animals to which saidcells are transplanted, animals in which immunological functions aredecreased or lacking are preferred, and there can be mentioned nudemouse, SCID mouse, beige mouse, and nude rat.

[0252] Furthermore, the anti-tumor effects to be evaluated can beconfirmed by variation in the amount of human immunoglobulins in theserum, measurement of tumor volume and/or weight, variation in theweight of human Bence Jones proteins in the urine, the survival periodof animals, or the like.

[0253] Pharmaceutical compositions or therapeutic agents for myelomathat contain as an active ingredient the chimeric anti-HM 1.24 antibodyor the reshaped human anti-HM 1.24 antibody of the present invention canbe systematically or locally administered in a parenteral manner. Forexample, intravenous injection such as drip infusion, intramuscularinjection, intraperitoneal injection, or subcutaneous injection can beselected and the dosage regimen may be selected as appropriate dependingon the age and the medical conditions of the patients.

[0254] Effective dosage is selected from the range of 0.01 mg to 1000mg/kg body weight/dose. Alternatively, the dosage of 5 mg/body,preferably 50 to 100 mg/body, may be selected.

[0255] Pharmaceutical compositions or therapeutic agents for myelomathat contain as an active ingredient the chimeric anti-HM 1.24 antibodyor the reshaped human anti-HM 1.24 antibody of the present invention maycontain pharmaceutically acceptable carriers or additives depending onthe route of administration.

[0256] As examples of such carriers and additives, there may bementioned water, pharmaceutically acceptable organic solvents, collagen,polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodiumcarboxymethyl cellulose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, arabic gum, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, pharmaceutically acceptablesurfactants, and the like. Additives to be used may be selected from,but not limited to, the above or combinations thereof.

EXAMPLES

[0257] Next, the present invention will be explained more specifically.

Example 1 Cloning of cDNA Encodinq the V Region of a Mouse Anti-HM 1.24Antibody

[0258] 1. Isolation of Messenger RNA (mRNA)

[0259] Using Fast Track mRNA Isolation Kit Version 3.2 (manufactured byInvitrogen) according to the instruction attached thereto, mRNA wasisolated from 2 ×10⁸ hybridoma cells (FERM BP-5233) that produce a mouseanti-HM 1.24 antibody.

[0260] 2. Amplification of the Gene Encoding the Variable Region ofAntibody by the PCR method

[0261] PCR was carried out using the amplification Thermal Cycler(manufactured by Perkin Elmer Cetus).

[0262] 2-1. Amplification and Fragmentation of the Gene Encoding the Vregion of a Mouse L chain

[0263] From the mRNA thus isolated, single stranded cDNA was synthesizedusing the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(manufactured by Life Science) and used for PCR. As primers used forPCR, MKV (Mouse Kappa Variable) primers (Jones, S. T. et al,Bio/Technology, 9, 88-89, (1991)) shown in SEQ ID NO: 29 to 39 thathybridize with the leader sequence of a mouse kappa type L chain wasused.

[0264] 100 μl of the PCR solution containing 10 mM Tris-HCl (pH 8.3), 50mM KCl, 0.1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl₂, 5 units ofDNA polymerase Ampli Taq (manufactured by Perkin Elmer Cetus), 0.25 mMof the MKV primers shown in SEQ ID NO: 29 to 39, 3 mM of the MKC primershown in SEQ ID NO: 40, and 100 ng of single stranded cDNA was coveredwith 50 μl of mineral oil, and then heated at an initial temperature of94° C. for 3 minutes, and then at 94° C. for 1 minute, at 55° C. for 1minute and at 72° C. for 1 minute in this order. After repeating thiscycle for 30 times, the reaction mixture was incubated at 72° C. for 10minutes. The amplified DNA fragment was purified by the low meltingpoint agarose (manufactured by Sigma), and digested with XmaI(manufactured by New England Biolabs) and SalI (manufactured by TakaraShuzo) at 37° C.

[0265] 2-2. Amplification and Fragmentation of cDNA Encoding the VRegion of a Mouse H Chain

[0266] The gene encoding the V region of a mouse H chain was amplifiedby the 5′-RACE method (Rapid Amplification of CDNA ends; Frohman, M. A.et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002, (1988), Edwards, J.B. D. M., et al., Nucleic Acids Res., 19, 5227-5232, (1991)). After cDNAwas synthesized using primer P1 (SEQ ID NO: 41) that specificallyhybridizes with the constant region of mouse IgG2a, cDNA encoding the Vregion of a mouse H chain was amplified by the 5′-AmpliFINDER RACE KIT(manufactured by CLONTECH) using the primer MHC2a (SEQ ID NO: 42) thatspecifically hybridizes with the constant region of mouse IgG2a and theanchor primer (SEQ ID NO: 77) attached to the kit. The amplified DNAfragment was purified with the low melting point agarose (manufacturedby Sigma) and digested with EcoRI (manufactured by Takara Shuzo) andXmaI (manufactured by New England Biolabs) at 37° C.

[0267] 3. Linking and Transformation

[0268] The DNA fragment comprising the gene encoding the V region of themouse kappa type L chain prepared as above was ligated to the pUC19vector prepared by digesting with SalI and XmaI by reacting in areaction mixture containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM ATP, 50 mg/ml of polyethylene glycol (8000) and oneunit of T4 DNA ligase (manufactured by GIBCO-BRL) at 16° C. for 2.5hours. Similarly, the DNA fragment comprising the gene encoding the Vregion of the mouse H chain was reacted and ligated to pUC19 vectorprepared by digesting with EcoRI and XmaI at 16° C. for three hours.

[0269] Then 10 μl of the above ligation mixture was added to 50 μl ofthe competent cells of Escherichia coli DH5α, which was left on ice for30 minutes, at 42° C. for one minute, and again on ice for one minute.Subsequently 400 μl of 2xYT medium (Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989))was added thereto, incubated at 37° C. for one hour, and then the E.coli was plated on the 2xYT agar medium (Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Laboratory Press, (1989))containing 50 μg/ml of ampicillin, and then incubated overnight at 37°C. to obtain the E. coli transformant.

[0270] The transformant was cultured overnight at 37° C. in 10 ml of the2xYT medium containing 50 μg/ml of ampicillin, and then from thisculture plasmid DNA was prepared using the alkali method (MolecularCloning: A Laboratory Manual, Sambrook et al., Cold Spring HarborLaboratory Press, (1989)).

[0271] The plasmid thus obtained containing the gene encoding the Vregion of the mouse kappa type L chain derived from the hybridoma thatproduces the anti-HM 1.24 antibody was termed pUCHMVL9. The plasmidobtained in the above-mentioned method containing the gene encoding theV region of the mouse H chain derived from the hybridoma that producesthe anti-HM 1.24 antibody was termed pUCHMVHR16.

Example 2 Determination of the Base Sequence of DNA

[0272] The base sequence of the cDNA coding region in theabove-mentioned plasmid was determined using the automatic DNA sequencer(manufactured by Applied Biosystem Inc.) and Taq Dye Deoxy TerminatorCycle Sequencing Kit (manufactured by Applied Biosystem Inc.) in theprotocol indicated by the manufacturer.

[0273] The base sequence of the gene encoding the V region of the Lchain of the mouse anti-HM 1.24 antibody contained in the plasmidpUCHMVL9 is shown in SEQ ID NO: 1. The base sequence of the geneencoding the V region of the H chain of the mouse anti-HM 1.24 antibodycontained in the plasmid pUCHMVHR16 is shown in SEQ ID NO: 2.

Example 3 Determination of CDR

[0274] The overall structures of the V regions of an L chain and an Hchain have similarity with each other, in which four framework portionsare linked by three hypervariable regions, i.e. complementaritydetermining regions (CDR). The amino acid sequence of the framework arerelatively well conserved but variation in the amino acid sequence isextremely high (Kabat, E.A., et al., “Sequences of Proteins ofImmunological Interest”, US Dept. Health and Human Services, 1983).

[0275] Based on these facts, the amino acid sequence of the variableregion of the anti-HM 1.24 antibody was compared the amino acidsequences of antibodies in the database to investigate homology, and theCDR region was determined as shown in Table 5. TABLE 5 Plasmid SequenceNo. CDR(1) CDR(2) CDR(3) pUCHMVL9 3-5 24-34 50-56 89-97 pUCHMVHR16 6-831-35 50-66  99-109

Example 4 Confirmation of Expression of the Cloned cDNA (Construction ofthe Chimeric Anti-HM 1.24 Antibody)

[0276] 1. Construction of an Expression Vector

[0277] In order to construct the expression vector that expresses achimeric anti-HM 1.24 antibody, cDNA clones pUCHMVL9 and pUCHMVHR16encoding the V regions of the L chain and the H chain of the mouseanti-HM 1.24 antibody, respectively, were modified by the PCR method,and then introduced into the HEF expression vector (InternationalApplication Publication No. WO 92-19759).

[0278] The backward primer ONS-L722S (SEQ ID NO: 43) for the V region ofan L chain and the backward primer VHR16S (SEQ ID NO: 44) for the Vregion of an H chain were designed so that they hybridize to the DNAencoding the start of the leader sequence of the V region of each andthey have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol.,196, 947-950, (1987)) and the recognition site for HindIII restrictionenzyme. The forward primer VL9A (SEQ ID NO: 45) for the V region of an Lchain and the forward primer VHR16A (SEQ ID NO: 46) for the V region ofan H chain were designed so that they hybridize to the DNA sequenceencoding the end of the J region and they have a splice donor sequenceand the recognition site for BamHI restriction enzyme.

[0279] 100 μl of the PCR reaction mixture containing 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.1 mM dNTPs , 1.5 mM MgCl₂, 100 pmole each of eachprimer, 100 ng of template DNA (pUCHMVL9 or pUCHMVHR16), and 5 units ofAmpli Taq enzyme was covered with 50 μl of mineral oil, and then afterthe initial denaturation at 94° C., heated at 94° C. for 1 minute, at55° C. for 1 minute and at 72° C. for 1 minute for 30 cycles and finallyincubated at 72° C. for 10 minutes.

[0280] The PCR product was purified by the 1.5% low melting pointagarose gel, and digested with HindIII and BamHI, and then cloned toHEF-VL-gk for the V region of the L chain and to HEF-VH-gγ1 for the Vregion of the H chain. After determination of the DNA sequence, theplasmids containing the DNA fragment that contains the correct DNAsequence were termed HEF-1.24L-gk and HEF-1.24H-gγ1, respectively.

[0281] The regions encoding the respective variable region from theabove plasmids HEF-1.24L-gk and HEF-1-24H-gγ1 were digested withrestriction enzymes HindIII and BamHI to make restriction fragments,which were inserted to the HindIII site and the BamHI sites of plasmidvector pUC19 and they were termed pUC19-1.24L-gk and pUC19-1.24H-gγ1,respectively.

[0282]Escherichia coli containing respective plasmids pUC19-1.24L-gk andpUC19-1.24H-gγ1 were termed Escherichia coli DH5α (pUC19-1.24L-gk) andEscherichia coli DH5α (pUC19-1.24H-gγ1), and were internationallydeposited on Aug. 29,1996, with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under theaccession numbers FERM BP-5646 and FERM BP-5644, respectively, under theprovisions of the Budapest Treaty.

[0283] 2. Transfection into COS-7 Cells

[0284] In order to observe the transient expression of the chimericanti-HM 1.24 antibody, the above expression vectors were tested in theCOS-7 (ATCC CRL-1651) cells. HEF-1.24L-gk and HEF-1.24H-gγ1 werecotransformed into COS-7 cells by electroporation using the Gene Pulserinstrument (manufactured by BioRad). Each DNA (10 μg) was added to 0.8ml aliquots of 1×10⁷ cells/ml in PBS, and was subjected to pulses at1500 V and a capacity of 25 μF.

[0285] After the recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of the DMEM culture liquid(manufactured by GIBCO) containing 10% γ-globulin free bovine fetalserum. After incubation of 72 hours in the CO₂ incubator BNA120D(manufactured by TABAI), the culture supernatant was collected, and thecell debris were removed by centrifugation, which were used for thefollowing experiment.

[0286] 3. FCM Analysis

[0287] The antigen binding activity of the chimeric anti-HM 1.24antibody was investigated by FCM (flow cytometry) analysis using theKPMM2 cells. After 4.7×10⁵ KPMM2 cells (Japanese Unexamined PatentPublication (Kokai) No. 7-236475) were washed with PBS(−), 50 μl of theculture of COS-7 cells that produces the above-mentioned chimericanti-HM 1.24 antibody and 50 μl of FACS buffer (PBS(−) containing 2%bovine fetal serum and 0.1% sodium azide), or 5 μl of 500 μg/ml purifiedmouse anti-HM 1.24 antibody and 95 μl of the FACS buffer were added, andincubated on ice for one hour.

[0288] As a control, 50 μl of 2 μg/ml chimeric SK2 (InternationalApplication Publication No. WO 94-28159) and 50 μl of the FACS buffer,or 5 μl of 500 μg/ml purified mouse IgG2aK (UPC10) (manufactured byCAPPEL in stead of purified mouse anti-HM 1.24 antibody, and 95 μl ofFACS buffer were added, and similarly incubated. After washing with theFACS buffer, 100 μl of 25 μg/ml FITC conjugated goat anti-human antibody(GAH) (manufactured by CAPPEL) or 10 μg/ml FITC conjugated goatanti-mouse antibody (GAM) (manufactured by Becton Dickinson) were added,and incubated at a temperature of ice for 30 minutes. After washing withthe FACS buffer, it was suspended in one ml of the FACS buffer, andfluorescence intensity of each cell was measured by the FACScan(manufactured by Becton Dickinson).

[0289] As shown in FIG. 1, it was revealed that the chimeric anti-HM1.24 antibody bound to the KPMM2 cell because the peak of fluorescenceintensity shifted to the right in the chimeric anti-HM 1.24antibody-added cells as compared to the control similarly to the casewhere mouse anti-HM 1.24 antibody was added. This confirmed that thecloned cDNA encodes the variable region of the mouse anti-HM 1.24antibody.

Example 5 Establishment of the CHO Cell Line That Stably Produces aChimeric Anti-HM 1.24 Antibody

[0290] 1. Construction of an Expression Vector for the Chimeric H Chain

[0291] After digesting the above plasmid HEF-1.24H-gγ1 with therestriction enzymes PvuI and BamHI, an about 2.8 kbp fragment containingthe EFI promoter and the DNA encoding-the V region of the H chain of themouse anti-HM 1.24 antibody was purified using 1.5% low melting pointagarose gel. Then, the above DNA fragment was inserted into an about 6kbp fragment prepared by digesting the expression vector used for ahuman H chain expression vector, DHFR-ΔE-Rvh-PM1f (see InternationalApplication Publication No. WO 92/19759), containing the DHFR gene andthe gene encoding the constant region of a human H chain with PvuI andBamHI to construct an expression vector, DHFR-ΔE-HEF-1.24H-gγ1, for theH chain of the chimeric anti-HM 1.24 antibody.

[0292] 2. Gene Introduction into CHO Cells

[0293] In order to establish a stable production system of the chimericanti-HM 1.24 antibody, the genes of the above-mentioned expressionvectors, HEF-1.24L-gk and DHFR-ΔE-HEF-1.24H-gγ1, that were linearized bydigestion with PvuI were simultaneously introduced into the CHO cellDXB11 (donated from the Medical Research Council Collaboration Center)by the electroporation method under the condition similar to theabove-mentioned one (the above-mentioned transfection into the COS-7cells).

[0294] 3. Gene Amplification by MTX

[0295] Among the gene-introduced CHO cells, only those CHO cells inwhich both of the L chain and the H chain expression vectors have beenintroduced can survive in the nucleoside-free α-MEM culture liquid(manufactured by GIBCO-BRL) to which 500 μg/ml G418 (manufactured byGIBCO-BRL) and 10% bovine fetal serum were added, and so they wereselected. Subsequently, 10 nM MTX (manufactured by Sigma) was added tothe above culture liquid. Among the clones that propagated, those thatproduce the chimeric anti-HM 1.24 antibody in large amounts wereselected. As a result, clones #8-13 that exhibit a production efficiencyof about 20 μg/ml of the chimeric antibody were obtained and termed thechimeric anti-HM 1.24 antibody-producing cell lines.

Example 6 Construction of the Chimeric Anti-HM 1.24 Antibody

[0296] The chimeric anti-HM 1.24 antibody was constructed in thefollowing method. The above chimeric anti-HM 1.24 antibody-producing CHOcells were subjected to continuous culture for 30 days using as themedium Iscove's Modified Dulbecco's Medium (manufactured by GIBCO-BRL)containing 5% γ-globulin free newborn bovine serum (manufactured byGIBCO-BRL) by the high-density cell culture instrument Verax system 20(manufactured by CELLEX BIOSCIENCE Inc.).

[0297] On day 13, 20, 23, 26, and 30 after starting the culture, theculture liquid was recovered using a pressurized filter unit SARTOBRAN(manufactured by Sartorius), and then the chimeric anti-HM 1.24 antibodywas affinity-purified using a large-volume antibody collection systemAfi-Prep System (manufactured by Nippon Gaishi) and Super Protein Acolumn (bed volume: 100 ml, manufactured by Nippon Gaishi) using PBS asthe absorption/wash buffer and 0.1 M sodium citrate buffer (pH 3) as theelution buffer according to the attached instructions. The elutedfractions were adjusted to about pH 7.4 by immediately adding 1 MTris-HCl (pH 8.0). Antibody concentration was measured by absorbance at280 nm and calculated with 1 μg/ml as 1.35 OD.

Example 7 Determination of Activity of the Chimeric Anti-HM 1.24Antibody

[0298] Chimeric anti-HM 1.24 antibody was evaluated by the followingbinding inhibition activity.

[0299] 1. Measurement of Binding Inhibition Activity

[0300] 1-1. Construction of a Biotinylated anti-HM 1.24 Antibody

[0301] After the mouse anti-HM 1.24 antibody was diluted with 0.1 Mbicarbonate buffer to 4 mg/ml, 4 μl of 50 mg/ml Biotin-N-hydroxysuccinimide (manufactured by EY LABS Inc.) was added and reacted at roomtemperature for 3 hours. Thereafter, 1.5 ml of 0.2 M glycine solutionwas added thereto, incubated at room temperature for 30 minutes to stopthe reaction, and then the biotinylated IgG fractions were collectedusing the PD-10 column (manufactured by Pharmacia Biotech).

[0302] 1-2. Measurement of Binding Inhibition Activity

[0303] The binding inhibition activity by the biotinylated mouse anti-HM1.24 antibody was measured by the Cell-ELISA using the human amnioticmembrane cell line WISH cells (ATCC CCL 25). The Cell-ELISA plates wereprepared as follows. To a 96-well plate was added 4 ×10⁵ cells/mlprepared with PRMI 1640 medium supplemented with 10% fetal bovine serum,incubated overnight, and after washing twice with PBS(−), wereimmobilized with 0.1% glutaraldehyde (manufactured by Nakalai tesque).

[0304] After blocking, 50 μl of serial dilutions of the chimeric anti-HM1.24 antibody or the mouse anti-HM 1.24 antibody obtained byaffinity-purification was added to each well and simultaneously 50 μl of2 μg/ml biotinylated mouse anti-HM 1.24 antibody was added, incubated atroom temperature for two hours, and then the peroxidase-labelledstreptavidin (manufactured by DAKO) was added. After incubating at roomtemperature for one hour and then washing, the substrate solution wasadded. After stopping the reaction by adding 50 μl of 6N sulfuric acid,absorbance at 490 nm was measured using the MICROPLATE READER Model 3550(manufactured by Bio-Rad).

[0305] The result, as shown in FIG. 2, revealed that the chimericanti-HM 1.24 antibody has the identical binding inhibition activity withthe mouse anti-HM 1.24 antibody to the biotinylated mouse anti-HM 1.24antibody. This indicates that the chimeric antibody had the same Vregion as the mouse anti-HM 1.24 antibody.

Example 8 Measurement of the ADCC Activity of the Chimeric anti-HM 1.24Antibody

[0306] ADCC (Antibody-dependent Cellular Cytotoxicity) activity wasmeasured according to the method as set forth in Current Protocols inImmunology, Chapter 7. Immunologic studies in humans, Editor, John E,Coligan et al., John Wiley & Sons, Inc., 1993.

[0307]1. Preparation of Effector Cells

[0308] Monocytes were separated from the peripheral blood or bone marrowof healthy humans and patients with multiple myeloma by the densitycentrifugation method. Thus, an equal amount of PBS(−) was added to theperipheral blood and the bone marrow of healthy humans and patients withmultiple myeloma, which was layered on Ficoll (manufactured byPharmacia)-Conrey (manufactured by Daiichi Pharmaceutical Co. Ltd.)(specific gravity, 1.077), and was centrifuged at 400 g for 30 minutes.The, monocyte layer was collected, and washed twice with RPMI 1640(manufactured by Sigma) supplemented with 10% fetal bovine serum(manufactured by Witaker), and prepared at a cell density of 5×10⁶/mlwith the same culture liquid.

[0309] 2. Preparation of Target Cells

[0310] The human myeloma cell line RPMI 8226 (ATCC CCL 155) wasradiolabelled by incubating in the RPMI 1640 (manufactured by Sigma)supplemented with 10% fetal bovine serum (manufactured by Witaker)together with 0.1 mCi of 51Cr-sodium chromate at 37° C. for 60 minutes.After radiolabelling, cells were washed three times with Hanks balancedsalt solution (HBSS) and adjusted to a concentration of 2×10⁵/ml.

[0311] 3. ADCC Assay

[0312] Into a 96-well U-bottomed plate (manufactured by Corning) wereadded 50 μl of 2×10⁵ target cells/ml, 1 μg/ml of affinity-purifiedchimeric anti-HM 1.24 antibody and mouse anti-HM 1.24 antibody, orcontrol human IgG (manufactured by Serotec), and reacted at 4° C. for 15minutes.

[0313] Then, 100 μl of 5×10⁶ effector cells/ml was added thereto, andcultured in the CO₂ incubator for 4 hours, when the ratio (E:T) of theeffector cells (E) to the target cells (T) was set at 0:1, 5:1, 20:1, or50:1.

[0314] One hundred μl of the supernatant was taken and the radioactivityreleased into the culture supernatant was measured by the gamma counter(ARC361, manufactured by Aloka). For measurement of the maximumradioactivity, 1% NP-40 (manufactured by BRL) was used. Cytotoxicity (%)was calculated by (A−C)/(B−C)×100, wherein A is radioactivity (cpm)released in the presence of antibody, B is radioactivity (cpm) releasedby NP-40, and C is radioactivity (cpm) released by the culture liquidalone without antibody.

[0315] As shown in FIG. 3, when the chimeric anti-HM 1.24 antibody wasadded as compared to the control IgG1, cytotoxicity increased with theincrease in the E:T ratio, which indicated that this chimeric anti-HM1.24 antibody has ADCC activity. Furthermore, since there was nocytotoxicity observed even when the mouse anti-HM 1.24 antibody wasadded, it was shown that the Fc portion of human antibody is required toobtain ADCC activity when the effector cell is a human-derived cell.

Example 9 Construction of the Reshaped Human Anti-HM 1.24 Antibody

[0316] 1. Designing of the V Region of the Reshaped Human Anti-HM 1.24Antibody

[0317] In order to construct the reshaped human antibody in which theCDR of mouse monoclonal antibody has been grafted to a human antibody,it is preferred that there is a high homology between the FR of themouse antibody and the FR of the human antibody. Thus, the V regions ofthe L chain and the H chain of the mouse anti-HM 1.24 antibody werecompared to the V regions of all known antibodies whose structure hasbeen elucidated using the Protein Data Bank.

[0318] The V region of the L chain of the mouse anti-HM 1.24 antibody ismost similar to the consensus sequence of the subgroup IV (HSGIV) of theV region of a human L chain with a homology of 66.4%. On the other hand,It has shown a homology of 56.9%, 55.8%, and 61.5% with HSGI, HSGII andHSG III, respectively.

[0319] When the V region of the L chain of the mouse anti-HM 1.24antibody is compared to the V region of the L chain of known humanantibodies, it has shown a homology of 67.0% with the V region REI of ahuman L chain, one of the subgroup I of the V region of a human L chain.Thus, the FR of REI was used as the starting material for constructionof the V region of the L chain of the reshaped human anti-HM 1.24antibody.

[0320] Version a of the V region of the L chain of the reshaped humananti-HM 1.24 antibody was designed. In this version, human FR was madeidentical with the REI-based FR present in the reshaped human CAMPATH-1Hantibody (see Riechmann, L. et al., Nature 322, 21-25, (1988), the FRcontained in version a of the V region of the L chain of the reshapedhuman PM-1 described in International Application Publication No. WO92-19759), and the mouse CDR was made identical with the CDR in the Vregion of the L chain of the mouse anti-HM 1.24 antibody.

[0321] The H chain V region of the mouse anti-HM 1.24 antibody is mostsimilar to the consensus sequence of HSGI of the V region of a human Hchain with a homology of 54.7%. On the other hand, it shows a homologyof 34.6% and 48.1% with HSGII and HSGIII, respectively. When the Vregion of the H chain of the mouse anti-HM 1.24 antibody is compared tothe V region of the H chain of known human antibodies, FR1 to FR3 weremost similar to the V region of the H chain of the human antibody HG3,one of subgroup I of the V region of a human H chain (Rechavi, G. etal., Proc. Natl. Acad. Sci. USA, 80, 855-859), with a homology of 67.3%.

[0322] Therefore, the FR of the human antibody HG3 was used as thestarting material for construction of the V region of the H chain of thereshaped human anti-HM 1.24 antibody; However, since the amino acidsequence of the FR4 of human HG3 has not been described, the amino acidsequence of the FR4 of the human antibody JH6 (Ravetch, J.V. et al.,Cell, 27, 583-591) that shows the highest homology with the FR4 of the Hchain of the mouse anti-HM 1.24 antibody was used. The FR4 of JH6 hasthe same amino acid sequence as that of the FR4 of the H chain of themouse anti-HM 1.24 antibody except one amino acid.

[0323] In the first version a of the V region of the H chain of thereshaped human anti-HM 1.24 antibody, FR1 to FR3 were made identicalwith the FR1 to FR3 of human HG3, and the CDR was made identical withthe CDR of the V region of the H chain of the mouse anti-HM 1.24antibody, except that the amino acids at position 30 in the human FR1and position 71 in the human FR3 were made identical with the aminoacids in the mouse anti-HM 1.24 antibody.

[0324] 2. Construction of the V Region of the L chain of the ReshapedHuman anti-HM 1.24 Antibody

[0325] The L chain of the reshaped human anti-HM 1.24 antibody wasconstructed by the CDR grafting in the PCR method. The method is shownin FIG. 4. Eight PCR primers were used for construction of the reshapedhuman anti-HM 1.24 antibody (version a) having the FR derived from thehuman antibody REI. The external primers A (SEQ ID NO: 47) and H (SEQ IDNO: 48) were designed to hybridize with the DNA sequence of theexpression vector HEF-VL-gk.

[0326] The CDR grafting primers L1S (SEQ ID NO: 49), L2S (SEQ ID NO:50), and L3S (SEQ ID NO: 51) have the sense DNA sequence. The CDRgrafting primers L1A (SEQ ID NO: 52), L2A (SEQ ID NO: 53), and L3A (SEQID NO: 54) have the antisense DNA sequence, each having a complementaryDNA sequence (20 to 23 bp) to the DNA sequence at the 5′-end of theprimers L1S, L2S, and L3S, respectively.

[0327] In the first stage of PCR, the four reactions A-L1A, L1S-L2A,L2S-L3A, and L3S-H were conducted to purify each PCR product. The fourPCR products from the first PCR were allowed to assemble with oneanother by their own complementarity (see International ApplicationPublication No. WO 92-19759). Then, external primers A and H were addedto amplify the full-length DNA encoding the V region of the L chain ofthe reshaped human anti-HM 1.24 antibody (the second PCR). In theabove-mentioned PCR, the plasmid HEF-RVL-M21a (see InternationalApplication Publication No. WO 95-14041) encoding the version a of the Vregion of the L chain of the reshaped human ONS-M21 antibody based onthe human antibody REI-derived FR was employed as a template.

[0328] In the first stage of PCR, the PCR mixture containing 10 mMTris-HCl (pH 8.3), 50 mM KCl, 0.1 mM dNTPs, 1.5 mM MgCl₂, 100 ng oftemplate DNA, 100 pmole of each primer, and 1.5 u of Ampli Taq was used.Each PCR tube was covered with 50 μl of mineral oil. Then after it wasfirst denatured by heating at 94° C., it was subjected to a reactioncycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1minute, and then was incubated at 72° C. for 10 minutes.

[0329] PCR products A-L1A (215 bp), L1S-L2A(98 bp), L2-L3A (140 bp), andL3S-H (151 bp) were purified using 1.5% low melting point agarose geland were assembled in the second PCR. In the second PCR, 98 μl of PCRmixture containing 1 μg each of the first stage PCR products and 5 u ofAmpli Taq was incubated for 2 cycles of 94° C. for 2 minutes, 55° C. for2 minutes, and 72° C. for 2 minutes, and then 100 pmole each of theexternal primers (A and H) was added. The PCR tube was coated with 50 μlof mineral oil and 30 cycles of PCR were conducted under the samecondition as above.

[0330] A 516 bp DNA fragment resulting from the second PCR was purifiedusing 1.5% low melting point agarose gel, digested with BamHI andHindIII, and the DNA fragments thus obtained were cloned into the HEFexpression vector HEF-VL-gk. After determining the DNA sequence, theplasmid containing the DNA fragment having the correct amino acidsequence of the V region of the L chain of the reshaped human anti-HM1.24 antibody was termed plasmid HEF-RVLa-AHM-gk. The amino acidsequence and the base sequence of the V region of L chain contained inthis plasmid HEF-RVLa-AHM-gk are shown in SEQ ID NO: 9.

[0331] The version b of the V region of the L chain of the reshapedhuman anti-HM 1.24 antibody was constructed by mutagenesis using PCR.Mutagen primers FTY-1 (SEQ ID NO: 55) and FTY-2 (SEQ ID NO: 56) were sodesigned as to mutate phenylalanine at position 71 to tyrosine.

[0332] After the above primers were amplified using the plasmidHEF-RVLa-AHM-gk as a template, the final product was purified anddigested with BamHI and HindIII. The DNA fragments obtained were clonedinto the HEF expression vector HEF-VL-gk to obtain plasmidHEF-RVLb-AHM-gk. The amino acid sequence and the base sequence of the Vregion of the L chain contained in this plasmid HEF-RVLb-AHM-gk areshown in SEQ ID NO: 10.

[0333]3. Construction of the V Region of the H Chain of the ReshapedhHman anti-HM 1.24 antibody 3-1. Construction of versions a to e of theV region of the H chain of the reshaped human anti-HM 1.24 antibody DNAencoding the V region of the H chain of the reshaped human anti-HM 1.24antibody was designed as follows. By linking the DNA sequence encodingthe FR1 to 3 of the human antibody HG3 and the FR4 of the human antibodyJH6 to the DNA sequence encoding the CDR of the V region of the H chainof the mouse anti-HM 1.24 antibody, the full length DNA encoding the Vregion of the H chain of the reshaped human anti-HM 1.24 antibody wasdesigned.

[0334] Then, to the 5′-end and the 3′-end of this DNA sequence theHindIII recognition site/KOZAK consensus sequence and BamHI recognitionsite/splice donor sequence, respectively, were attached so as to enableinsertion of the HEF expression vector.

[0335] The DNA sequence thus designed was divided into fouroligonucleotides. Subsequently, oligonucleotides which potentiallyhinder assembly of these oligonucleotides were subjected to computeranalysis for the secondary structure. The sequences of the fouroligonucleotides RVH1 to RVH4 are shown in SEQ ID NO: 57 to 60. Theseoligonucleotides have a length of 119 to 144 bases and have the 25 to 26bp overlapping region. Among the oligonucleotides, RVH2 (SEQ ID NO: 58)and RVH4 (SEQ ID NO: 60) have the sense DNA sequence, and RVH1 (SEQ IDNO: 57) and RVH3 (SEQ ID NO: 59) have the antisense DNA sequence. Themethod for assembling these four oligonucleotides by the PCR method isshown in the figure (see FIG. 5).

[0336] The PCR mixture (98 μl) containing 100 ng each of the fouroligonucleotides and 5 u of Ampli Taq was first denatured by heating at94° C. for 2 minutes, and was subjected to two cycles of incubationcomprising 94° C. for 2 minutes, 55° C. for 2 minutes and 72° C. for 2minutes. After 100 pmole each of RHP1 (SEQ ID NO: 61) and RHP2 (SEQ IDNO: 62) were added as the external primer, the PCR tube was coated with50 μl of mineral oil. Then it was first denatured by heating at 94° C.for 1 minute, and then was subjected to 38 cycles of 94° C. for 1minute, 55° C. for 1 minute and 72° C. for 1 minute, and then wasincubated at 72° C. for 10 minutes.

[0337] The 438 bp DNA fragment was purified using 1.5% low melting pointagarose gel, digested with HindIII and BamHI, and then cloned into theHEF expression vector HEF-VH-gγ1. After determination of the basesequence, the plasmid that contains the DNA fragment encoding the aminoacid sequence of the correct V region of the H chain was termedHEF-RVHa-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHa-AHM-gγ1 areshown in SEQ ID NO: 11.

[0338] Each of versions b, c, d, and e of the V region of the H chain ofthe reshaped human anti-HM 1.24 antibody was constructed as follows.

[0339] Using as the mutagen primer BS (SEQ ID NO: 63) and BA (SEQ ID NO:64) designed to mutate arginine at position 66 to lysine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version bwas amplified to obtain plasmid HEF-RVHb-AHM-gγ. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHb-AHM-gγ1 are shown in SEQ ID NO: 12.

[0340] Using as the mutagen primer CS (SEQ ID NO: 65) and CA (SEQ ID NO:66) designed to mutate threonine at position 73 to lysine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1 by the PCR method, version cwas amplified to obtain plasmid HEF-RVHc-AHM-gγ1. The amino acidsequence and the base sequence of the V region of the H chain containedin this plasmid HEF-RVHc-AHM-gγ2are shown in SEQ ID NO: 13.

[0341] Using as the mutagen primer DS (SEQ ID NO: 67) and DA (SEQ ID NO:68) designed to mutate arginine at position 66 to lysine and threonineat position 73 to lysine and as a template DNA the plasmidHEF-RVHa-AHM-gγ1 by the PCR method, version d was amplified to obtainplasmid HEF-RVHd-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHd-AHM-gγ1 are shown in SEQ ID NO: 14.

[0342] Using as the mutagen primer ES (SEQ ID NO: 69) and EA (SEQ ID NO:70) designed to mutate valine at position 67 to alanine and methionineat position 69 to leucine and as a template DNA the plasmidHEF-RVHa-AHM-gγ1, version e was amplified to obtain plasmidHEF-RVHe-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHe-AHM-gγ1 areshown in SEQ ID NO: 15.

[0343]3-2. Construction of the H Chain Hybrid V Region

[0344] Two H chain hybrid V regions were constructed. One is a mousehuman hybrid anti-HM 1.24 antibody in which the amino acid sequences ofFR1 and FR2 are derived from the mouse anti-HM 1.24 antibody and thoseof FR3 and FR4 are from version a of the V region of the H chain of thereshaped human anti-HM 1.24 antibody, and the other is human mousehybrid anti-HM 1.24 antibody in which the amino acid sequences of FR1and FR2 are derived from version a of the V region of the H chain of thereshaped human anti-HM 1.24 antibody and those of FR3 and FR4 are fromthe mouse anti-HM 1.24 antibody. The amino acid sequences of the CDRregions are all derived from mouse anti-HM 1.24 antibody.

[0345] Two H chain hybrid V regions were constructed by the PCR method.The method is schematically shown in FIG. 6 and 7. For the constructionof two H chain hybrid V regions, four primers were used. The externalprimers a (SEQ ID NO: 71) and h (SEQ ID NO: 72) were designed tohybridize with the DNA sequence of the HEF expression vector HEF-VH-gγ1.The H chain hybrid construction primer HYS (SEQ ID NO: 73) was designedto have the sense DNA sequence and the H chain hybrid primer HYA (SEQ IDNO: 74) to have the antisense DNA sequence so that the DNA sequence arecomplementary to each other.

[0346] For the construction of the H chain hybrid V region in which theamino acid sequences of FR1 and FR2 are derived from the mouse anti-HM1.24 antibody and those of FR3 and FR4 are from version a of the Vregion of the H chain of the reshaped human anti-HM 1.24 antibody, PCRusing the plasmid HEF-1.24H-gγ1 as a template, the external primer a,and the H chain hybrid primer HYA, and PCR using the plasmidHEF-RVHa-AHM-gγ1 as a template, the H chain hybrid primer HYS (SEQ IDNO: 73), and the external primer h (SEQ ID NO: 72) were carried out inthe first stage of PCR and each PCR product was purified. The two PCRproducts from the first PCR were allowed to assemble by their owncomplementarity (see International Application Publication No. WO92-19759).

[0347] Then, by adding the external primers a (SEQ ID NO: 71) and h (SEQID NO: 72) a full-length DNA encoding the H chain hybrid V region inwhich the amino acid sequences of FR1 and FR2 are derived from the mouseanti-HM 1.24 antibody and those of FR3 and FR4 are from version a of theV region of the H chain of the reshaped human anti-HM 1.24 antibody wasamplified in the second PCR stage.

[0348] For the construction of the H chain hybrid V region in which theamino acid sequences of FRI and FR2 are derived from version a of the Vregion of the H chain of the reshaped human anti-HM 1.24 antibody andthose of FR3 and FR4 are from the mouse anti-HM 1.24 antibody, PCR usingthe plasmid HEF-RVHa-AHM-gγ1 as a template, the external primer a, andthe H chain hybrid primer HYA, and PCR using the plasmid HEF-1.24H-gγ1as a template, the H chain hybrid primer HYS, and the external primer hwere carried out in the first stage of PCR and each PCR product waspurified. The two PCR purified products from the first PCR were allowedto assemble by their own complementarity (see International ApplicationPublication No. WO 92-19759).

[0349] Then, by adding the external primers a and h, a full-length DNAencoding the H chain hybrid V region in which the amino acid sequencesof FR1 and FR2 are derived from version a of the V region of the H chainof the reshaped human anti-HM 1.24 antibody and those of FR3 and FR4 arefrom the mouse anti-HM 1.24 antibody was amplified in the second PCRstage.

[0350] The methods of the first PCR, purification of PCR products,assembling, the second PCR, and cloning into the HEF expression vectorHEF-VH-gγ1 were carried out according to the methods shown in “Example9. Construction of the V region of the L chain of the reshaped humananti-HM 1.24 antibody”.

[0351] After determination of the DNA sequence, the plasmid thatcontains the DNA fragment encoding the correct amino acid sequence ofthe H chain hybrid V region in which the amino acid sequences of FR1 andFR2 are derived from the mouse anti-HM 1.24 antibody and those of FR3and FR4 are from version a of the V region of the H chain of thereshaped human anti-HM 1.24 antibody was termed HEF-MH-RVH-AHM-gγ1. Theamino acid sequence and the base sequence of the V region of the H chaincontained in this plasmid HEF-MH-RVH-AHM-gγ1 are shown in SEQ ID NO: 75.Also, the plasmid that contains the DNA fragment encoding the correctamino acid sequence of the H chain hybrid V region in which the aminoacid sequences of FR1 and FR2 are derived from version a of the V regionof the H chain of the reshaped human anti-HM 1.24 antibody and those ofFR3 and FR4 are from the mouse anti-HM 1.24 antibody was termedHEF-HM-RVH-AHM-gγ1. The amino acid sequence and the base sequence of theV region of the H chain contained in this plasmid HEF-HM-RVH-AHM-gγ1 areshown in SEQ ID NO: 76.

[0352]3-3. Construction of Versions f to s of the V region of the Hchain of the reshaped human anti-HM 1.24 antibody

[0353] Each of versions f, g, h, i, j, k, l, m, n, o, p, q, r, and s ofthe V region of the H chain of the reshaped human anti-HM 1.24 antibodywere constructed as follows.

[0354] Using as the mutagen primer FS (SEQ ID NO: 78) and FA (SEQ ID NO:79) designed to mutate threonine at position 75 to serine and valine atposition 78 to alanine and as a template DNA the plasmidHEF-RVHe-AHM-gγ1 by the PCR method, version f was amplified to obtainplasmid HEF-RVHf-AHM-gγ1. The amino acid sequence and the base sequenceof the V region of the H chain contained in this plasmidHEF-RVHf-AHM-gγ1 are shown in SEQ ID NO: 16.

[0355] Using as the mutagen primer GS (SEQ ID NO: 80) and GA (SEQ ID NO:81) designed to mutate alanine at position 40 to arginine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version g was amplified toobtain plasmid HEF-RVHg-AHM-gγ1.The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHg-AHM-gγl are shown in SEQ ID NO: 17.

[0356] Using as the mutagen primer FS (SEQ.ID NO: 78) and FA (SEQ ID NO:79) and as a template DNA the plasmid HEF-RVHb-AHM-gγ1, version h wasamplified to obtain plasmid HEF-RVHh-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHh-AHM-gγ1 are shown in SEQ ID NO: 18.

[0357] Using as the mutagen primer IS (SEQ ID NO: 82) and IA (SEQ ID NO:83) designed to mutate arginine at position 83 to alanine and serine atposition 84 to phenylalanine as a template DNA the plasmidHEF-RVHh-AHM-gγ1, version i was amplified to obtain plasmidHEF-RVHi-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHi-AHM-gγ1 areshown in SEQ ID NO: 19.

[0358] Using as the mutagen primer JS (SEQ ID NO: 84) and JA (SEQ ID NO:85) designed to mutate arginine at position 66 to lysine and as atemplate DNA the plasmid HEF-RVHf-AHM-gγ1, version j was amplified toobtain plasmid HEF-RVHj-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHj-AHM-gγ1 are shown in SEQ ID NO: 20.

[0359] Using as the mutagen primer KS (SEQ ID NO: 86) and KA (SEQ ID NO:87) designed to mutate glutamic acid at position 81 to glutamine and asa template DNA the plasmid HEF-RVHh-AHM-gγ1, version k was amplified toobtain plasmid HEF-RVHk-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHk-AHM-gγ1 are shown in SEQ ID NO: 21.

[0360] Using as the mutagen primer LS (SEQ ID NO: 88) and LA (SEQ ID NO:89) designed to mutate glutamic acid at position 81 to glutamine andserine at position 82B to isoleucine and as a template DNA the plasmidHEF-RVHh-AHM-gγ1, version 1 was amplified to obtain plasmidHEF-RVHl-AHM-gγ1. The amino acid sequence and the base sequence of the Vregion of the H chain contained in this plasmid HEF-RVHl-AHM-gγ1 areshown in SEQ ID NO: 22.

[0361] Using as the mutagen primer MS (SEQ ID NO: 90) and MA (SEQ ID NO:91) designed to mutate glutamic acid at position 81 to glutamine, serineat position 82b to isoleucine, and threonine at position 87 to serineand as a template DNA the plasmid HEF-RVHh-AHM-gγ1, version m wasamplified to obtain plasmid HEF-RVHm-AHM-gγ1. The amino acid sequenceand the base sequence of the V region of the H chain contained in thisplasmid HEF-RVHm-AHM-gγ1are shown in SEQ ID NO: 23.

[0362] Using as the mutagen primer NS (SEQ ID NO: 92) and NA (SEQ ID NO:93) designed to mutate serine at position 82B to isoleucine and as atemplate DNA the plasmid HEF-RVHh-AHM-gγ1, version n was amplified toobtain plasmid HEF-RVHn-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHn-AHM-gγ1 are shown in SEQ ID NO: 24.

[0363] Using as the mutagen primer OS (SEQ ID NO: 94) and OA (SEQ ID NO:95) designed to mutate threonine at position 87 to serine and as atemplate DNA the plasmid HEF-RVHh-AHM-gγ1, version o was amplified toobtain plasmid HEF-RVHo-AHM-gγ1. The amino acid sequence and the basesequence of the V region of the H chain contained in this plasmidHEF-RVHo-AHM-gγ1 are shown in SEQ ID NO: 25.

[0364] Using as the mutagen primer PS (SEQ ID NO: 96) and PA (SEQ ID NO:97) designed to mutate valine at position 78 to alanine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version p was amplified bythe PCR method to obtain plasmid HEF-RVHp-AHM-gγ1. The amino acidsequence and the base sequence of the V region of the H chain containedin this plasmid HEF-RVHp-AHM-gγ1 are shown in SEQ ID NO: 26.

[0365] Using as the mutagen primer QS (SEQ. ID NO: 98) and QA (SEQ IDNO: 99) designed to mutate threonine at position 75 to serine and as atemplate DNA the plasmid HEF-RVHa-AHM-gγ1, version q was amplified bythe PCR method to obtain plasmid HEF-RVHq-AHM-gγ1. The amino acidsequence and the base sequence of the V region of the H chain containedin this plasmid HEF-RVHq-AHM-gγ1 are shown in SEQ ID NO: 27.

[0366] Using as the mutagen primer CS (SEQ ID NO: 65) and CA (SEQ ID NO:66) and as a template DNA the plasmid HEF-RVHp-AHM-gγ1, version r wasamplified by the PCR method to obtain plasmid HEF-RVHr-AHM-gγ1. Theamino acid sequence and the base sequence of the V region of the H chaincontained in this plasmid HEF-RVHr-AHM-gγ1 are shown in SEQ ID NO: 28.

[0367] Version s of the V region of the H chain of the reshaped humananti-HM 1.24 antibody was constructed by mutagenesis using PCR. Themutagen primers SS (SEQ ID NO: 100) and SA (SEQ ID NO: 101) weredesigned to mutate methionine at position 69 to isoleucine.

[0368] After the above primer was amplified using plasmidHEF-RVHr-AHM-gγ1 as a template, the final product was purified, digestedwith BamHI and HindIII, and the DNA fragment obtained was cloned intothe HEF expression vector HEF-VH-gγ1 to obtain plasmid HEF-RVHs-AHM-gγ1.The amino acid sequence and the base sequence of the V region of the Hchain contained in this plasmid HEF-RVHs-AHM-gγ1 are shown in SEQ ID NO:102.

[0369] The regions encoding the variable region of each of theabove-mentioned plasmids HEF-RVLa-AHM-gk and HEF-RVHr-AHM-gγ1 weredigested to make restriction fragments with restriction enzymes HindIIIand BamHI. They were inserted into the HindIII and BamHI sites ofplasmid vector pUC19. Each plasmid was termed pUC19-RVLa-AHM-gk andpUC19-RVHr-AHM-gγ1.

[0370] The Escherichia coli that contains each of the plasmidspUC19-RVLa-AHM-gK and pUC19-RVHr-AHM-gγ1 was termed Escherichia coliDH5a (pUC19-RVLa-AHM-gK) and Escherichia coli DH5α (pUC19-RVHr-AHM-gγ1),respectively, and has been internationally deposited on Aug. 29,1996,with the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, MITI (Higashi 1-Chome 1-3, Tsukubacity, Ibalaki prefecture, Japan) under the accession number FERM BP-5645and FERM BP-5643, respectively, under the provisions of the BudapestTreaty.

[0371] The regions encoding the variable region of the above-mentionedplasmid HEF-RVHs-AHM-gγ1 were digested to make a restriction fragmentwith restriction enzymes HindIII and BamHI. They were inserted into theHindIII and BamHI sites of plasmid vector pUC19. The plasmid obtainedwas termed pUC19-RVHs-AHM-gγ1.

[0372] The Escherichia coli that contains the plasmid pUC19-RVHs-AHM-gγ1was termed Escherichia coli DH5α ( (pUC19-RVHs-AHM-gγ1), and has beeninternationally deposited on Sep. 29,1997, with the National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology, MITI (Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture,Japan) under the accession number FERM BP-6127 under the provisions ofthe Budapest Treaty.

[0373] 4. Construction of the Reshaped Human Anti-HM 1.24 Antibody, thechimeric anti-HM 1.24 antibody, and the H chain hybrid antibody

[0374] In order to evaluate each chain of the reshaped human anti-HM1.24 antibody, the reshaped human anti-HM 1.24 antibody and the chimericanti-HM 1.24 antibody as a positive control antibody were allowed toexpress. In constructing each of version b and after of the V region ofthe H chain of the reshaped human anti-HM 1.24 antibody, the H chainhybrid antibody was allowed to express in order to investigate whichamino acid sequence in the FR should be substituted. Furthermore, it wasexpressed in combination with the chimeric H chain in order to evaluateversion a of L chain of the reshaped human anti-HM 1.24 antibody.

[0375] 4-1. Expression of the Reshaped Human Anti-HM 1.24 Antibody (1)

[0376] Ten μg each of the expression vector (HEF-RVHa-AHM-gγ1 toHEF-RVHr-AHM-gγ1) for the H chain of the reshaped human anti-HM 1.24antibody and the expression vector (HEF-RVLa-AHM-gk or HEF-RVLb-AHM-gk)for the L chain of the reshaped human anti-HM 1.24 antibody werecotransformed into COS-7 cells by electroporation using the Gene Pulserinstrument (manufactured by BioRad). Each DNA (10 μg) was added to 0.8ml aliquots of 1×10⁷ cells/ml in PBS, and was subjected to pulses at1500 V and a capacity of 25 μF.

[0377] After the recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of DMEM culture medium(manufactured by GIBCO) containing 10% γ-globulin free fetal bovineserum. After incubation of 72 hours in the CO₂ incubator BNA120D(manufactured by TABAI) under the condition of 37° C. and 5% CO₂, theculture supernatant was collected, the cell debris were removed bycentrifugation at 1000 rpm for 5 minutes in a centrifuge 15PR-22(manufactured by HITACHI) equipped with a centrifuge rotor 03(manufactured by HITACHI), and the microconcentrator (Centricon 100,manufactured by Amicon) was ultrafiltrated using a centrifuge J2-21(manufactured by BECKMAN) equipped with a centrifuge rotor JA-20.1(manufactured by BECKMAN), and was used for Cell-ELISA.

[0378] Expression of the Reshaped Human Anti-HM 1.24 aAntibody (2)

[0379] Ten μg each of the expression vector (HEF-RVHs-AHM-gγ1) forversion “s” of the H chain of the reshaped human anti-HM 1.24 antibodyand the expression vector (HEF-RVLa-AHM-gk) for the L chain of thereshaped human anti-HM 1.24 antibody were cotransformed into COS-7 cellsby electroporation using the Gene Pulser instrument (manufactured byBioRad). Each DNA (10 μg) was added to 0.8 ml aliquots of 1 x 10cells/ml in PBS, and was subjected to pulses at 1500 V and a capacity of25 pF.

[0380] After the recovery period of 10 minutes at room temperature, theelectroporated cells were added to 30 ml of DMEM culture medium(manufactured by GIBCO) containing 10% γ-globulin free fetal bovineserum. After incubation of 72 hours in the CO₂ incubator BNA120D(manufactured by TABAI) under the condition of 37° C. and 5% CO₂, theculture supernatant was collected, the cell debris were removed bycentrifugation at 1000 rpm for 5 minutes in a centrifuge 05PR-22(manufactured by HITACHI) equipped with a centrifuge rotor 03(manufactured by HITACHI), and the microconcentrator (Centricon 100,manufactured by Amicon) was concentrated by ultrafiltration using acentrifuge J2-21 (manufactured by BECKMAN) equipped with a centrifugerotor JA-20.1 (manufactured by BECKMAN), and was filtration-sterilizedusing a filter, Millex GV13mm (manufactured by Millipore), which wasused for Cell-ELISA.

[0381] 4-2. Expression of the Chimeric Anti-HM 1.24 Antibody

[0382] Using Ten μg each of the expression vector HEF-1.24H-gγ1 for theH chain of the chimeric anti-HM 1.24 antibody and the expression vectorHEF-1.24L-gλfor the L chain of the chimeric anti-HM 1.24 antibody, thechimeric anti-HM 1.24 antibody to be used for Cell-ELISA was preparedaccording to the above-mentioned method for expression of the reshapedhuman anti-HM 1.24 antibody.

[0383] 4-3. Expression of the Anti-HM 1.24 Antibody Comprising Version aof the Humanized L Chain and the Chimeric H Chain

[0384] Using Ten μg each of the expression vector HEF-1.24H-gγ1 for theH chain of the chimeric anti-HM 1.24 antibody and the expression vectorHEF-RyLa-AHM-gk for version a of the L chain of the reshaped humananti-HM 1.24 antibody, the anti-HM 1.24 antibody comprising version a ofthe humanized L chain and the chimeric H chain to be used for Cell-ELISAwas prepared according to the above-mentioned method for expression ofthe reshaped human anti-HM 1.24 antibody.

[0385] 4-4. Expression of the H Chain Hybrid Antibody

[0386] Using Ten μg each of the expression vector (HEF-MH-RVH-AHM-gγ1 orHEF-HM-RVH-AHM-gγ1) for the V region of the H chain hybrid and theexpression vector HEF-RVLa-AHM-gk for the L chain of the reshaped humananti-HM 1.24 antibody, the H chain hybrid antibody to be used forCell-ELISA was prepared according to the above-mentioned method forexpression of the reshaped human anti-HM 1.24 antibody. 4-5. Measurementof antibody concentration Concentration of the antibody obtained wasmeasured by ELISA. Each well of a 96-well ELISA plate (Maxisorp,manufactured by NUNC) was immobilized by adding 100 μl of goatanti-human IgG antibody (manufactured by BIO SOURCE) prepared to aconcentration of 1 μg/ml with the coating buffer (0.1 M NaHCO₃, 0.02%NaN₃, pH 9.6) and incubating at room temperature for one hour. Afterblocking with 100 μl of the dilution buffer (50 mI Tris-HCl, 1 mM MgCl₂,0.15 M NaCl, 0.05% Tween 20, 0.02% NaN₃, 1% bovine serum albumin (BSA),pH 8.1), 100 μl each of serial dilutions of the culture supernatant ofcos-7 cells secrating the reshaped human anti-HM 1.24 antibody, thechimeric anti-HM 1.24 antibody, or the H chain hybrid antibody that wereconcentrated by ultrafiltration were added to each well and incubated atroom temperature for one hour. Then after washing, 100 μl of alkalinephosphatase-labelled goat anti-human IgG antibody (manufactured by DAKO)was added.

[0387] After incubating at room temperature for one hour and washing,100 μl of 1 μg/ml substrate solution (Sigma104, p-nitrophenyl phosphate,SIGMA) dissolved in the substrate buffer (50 mM NaHCO₃10 MM MgCl₂₁ pH9.8) was added, and then the absorbance at 405 nm was measured using theMICROPLATE READER Model 3550 (manufactured by Bio Rad). As the standardfor the measurement of concentration, human IgG1k (manufactured by Thebinding Site) was used.

[0388] 5. Establishment of the CHO Cell Line that Stably Produces theReshaped Human Anti-HM 1.24 Antibody

[0389] 5-1. Construction of the Expression Vector for the H Chain of theReshaped Human Anti-HM 1.24 Antibody

[0390] By digesting plasmid HEF-RVHr-AHM-gγ1 with the restrictionenzymes PvuI and BamHI, an about 2.8 kbp fragment containing the DNAencoding the EF1 promoter and the V region of the H chain of thereshaped human anti-HM 1.24 antibody was purified using 1.5% low meltingpoint agarose gel. Then, the above DNA fragment was inserted into anabout 6 kbp fragment that was prepared by digesting the expressionvector used for a human H chain expression vector, DHFR-ΔE-RVh-PM1f(International Application Publication No. WO 92-19759), containing theDHFR gene and the gene encoding the constant region of a human H chainwith PvuI and BamHI to construct an expression vector,DHFR-ΔE-HEF-RVHr-AHM-gγ1, for the H chain of the reshaped anti-HM 1.24antibody.

[0391] 5-2. Gene Introduction into CHO Cells

[0392] In order to establish a stable production system of the reshapedhuman anti-HM 1.24 antibody, the genes of the above-mentioned expressionvectors, DHFR-ΔE-HEF-RVHr-AHM-gγ1 and HEF-RVLa-AHM-gk, that werelinearized by digestion with PvuI were simultaneously introduced intothe CHO cell DXB-11 by the electroporation method under the conditionsimilar to the above-mentioned one (transfection into theabove-mentioned COS-7 cells).

[0393] 5-3. Gene Amplification by MTX

[0394] Among the gene-introduced CHO cells, only those CHO cells inwhich both of L chain and H chain expression vectors have beenintroduced can survive in the nucleoside-free α-MEM culture medium(manufactured by GIBCO-BRL) to which 500 μg/ml G418 (manufactured byGIBCO-BRL) and 10% fetal bovine serum were added, and so they wereselected. Subsequently, 10 nM MTX (manufactured by Sigma) was added tothe above culture medium. Among the clones that propagated, those thatproduce the reshaped human anti-HM 1.24 antibody in large amounts wereselected. As a result, clone 1 that exhibits a production efficiency ofabout 3 μg/ml of the reshaped human anti-HM 1.24 antibody was obtainedand termed the reshaped human anti-HM 1.24 antibody-producing cell line.

[0395] 5-4. Construction of the Reshaped Human Anti-HM 1.24 Antibody

[0396] The reshaped anti-HM 1.24 antibody was constructed in thefollowing method. The above CHO cells that produce the reshaped humananti-HM 1.24 antibody were cultured for 10 days using as the medium thenucleoside-free α-MEM culture medium (manufactured by GIBCO-BRL) towhich 500 μg/ml G418 (manufactured by GIBCO-BRL) and 10% y-free fetalbovine serum were added using the CO₂ incubator BNAS120D (manufacturedby TABAI) under the condition of 37° C. and 5% CO₂. On day 8 and 10after starting the culture the culture liquid was recovered, the celldebris were removed by centrifuging for 10 minutes at 2000 rpm using thecentrifuge RL-500SP (manufactured by Tomy Seiko) equipped with the TS-9rotor, and then filter-sterilized using a bottle top filter(manufactured by FALCON) having a membrane of 0.45 μm in diameter.

[0397] After an equal amount of PBS(−) was added to the culture liquidof the CHO cells that produce the reshaped human anti-HM 1.24 antibody,then the reshaped human anti-HM 1.24 antibody was affinity-purifiedusing the high-speed antibody purification system ConSep LC100(manufactured by MILLIPORE) and Hyper D Protein A column (manufacturedby Nippon Gaishi) using PBS(−) as the absorption/wash buffer and 0.1 Msodium citrate buffer (pH 3) as the elution buffer according to theattached instructions. The eluted fractions were adjusted to about pH7.4 by immediately adding 1 M Tris-HCl (pH 8.0) and then using thecentrifuging ultrafiltration concentrator Centriprep-10 (manufactured byMILLIPORE), concentration and substitution to PBS(−) was carried out andfilter-sterilized using a membrane filter MILLEX-GV (manufactured byMILLIPORE) with a pore size of 0.22 μm to obtain the purified reshapedhuman anti-HM 1.24 antibody. Antibody concentration was measured byabsorbance at 280 nm and calculated with 1 mg/ml as 1.35 OD.

Example 11 Determination of Activity of the Reshaped Human Anti-HM 1.24Antibody

[0398] The reshaped human anti-HM 1.24 antibody was evaluated for thefollowing antigen binding activity and binding inhibition activity.

[0399] 1. The Method of Measurement of Antigen Binding Activity andBinding Inhibition Activity

[0400] 1-1; Measurement of Antigen Binding Activity

[0401] Antigen binding activity was measured by the Cell-ELISA usingWICH cells. Cell-ELISA plates were prepared as described in the aboveExample 7.1-2.

[0402] After blocking, 100 μl of serial dilutions of the reshaped humananti-HM 1.24 antibody that was obtained from the concentrate of theculture supernatant of COS-7 cells or purified from the culturesupernatant of CHO cells was added to each well. After it was incubatedfor 2 hours at room temperature and washed, peroxidase-labelled rabbitanti-human IgG antibody (manufactured by DAKO) was added. After it wasincubated for 1 hour at room temperature and washed, 100 μl of substratesolution was added in each well. After incubation, the reaction wasstopped by 50 μl of 6N sulfuric acid, and absorbance at 490 nm wasmeasured using the MICROPLATE READER Model 3550 (manufactured byBio-Rad).

[0403] 1-2. Measurement of Binding Inhibition Activity

[0404] The binding inhibition activity by the biotin-labelled mouseanti-HM 1.24 antibody was measured by the Cell-ELISA using WISH cells.Cell ELISA plates were prepared as described in the above Example 7.1-2. After blocking, 50 μl of serial dilutions of the reshaped humananti-HM 1.24 antibody that was obtained from the concentrate of theculture supernatant of COS-7 cells or purified from the culturesupernatant of CHO cells was added to each well, and 50 μl of thebiotin-labelled mouse anti-HM 1.24 antibody was added simultaneously.After incubating at room temperature for two hours and washing,peroxidase-labelled streptavidin (manufactured by DAKO) was added. Afterincubating at room temperature for one hour and then washing, 100 μl ofsubstrate solution was added in each well. After incubation, thereaction was stopped by 50 μl of 6N sulfuric acid, and absorbance at 490nm was measured using the MICROPLATE READER Model 3550 (manufactured byBio-Rad).

[0405] 2. Evaluation of the reshaped human anti-HM 1.24 antibody

[0406] 2-1. L chain

[0407] Version a of the L chain of the reshaped human anti-HM 1.24antibody was evaluated as mentioned for measurement of antigen bindingactivity. As shown in FIG. 8, when version a of the L chain is expressedin combination with the chimeric H chain it has shown a similar level ofantigen binding activity. However, in consideration of further increasein activity and of compatibility with the H chain, version b of the Lchain was constructed. Versions a and b of the L chain were evaluatedtogether for antigen binding activity and of binding inhibition activitywhen combined with versions a, b, f, or h of the H chain. As shown inFIG. 9, 10, 11, and 12, version a of the L chain had a higher activitythan version b in both activities in all versions a, b, f, and h of theH chain. Therefore, version a of the L chain of the reshaped humananti-HM 1.24 antibody was used for the following experiment.

[0408] 2-2. H Chain Versions a to e

[0409] Versions a to e of the H chain of the reshaped human anti-HM 1.24antibody were evaluated in combination with the version a of the L chainas mentioned for measurement of antigen binding activity and bindinginhibition activity. The result, as shown in FIG. 11, 13, 14, and 15,indicated that all versions were weaker in both activities as comparedto the chimeric anti-HM 1.24 antibody, suggesting that further aminoacid substitution is required.

[0410] 2-3. The H Chain Hybrid Antibody

[0411] The H chain hybrid antibody was evaluated as mentioned formeasurement of antigen binding activity. The result, as shown in FIG.16, indicated that the human-mouse hybrid anti-HM 1.24 antibody hasshown a similar activity to that of the chimeric anti-HM 1.24 antibodyfor antigen binding activity, whereas the mouse, human hybrid anti-HM1.24 antibody had a weaker activity than the chimeric anti-HM 1.24antibody. This indicated that in order to construct the reshaped humananti-HM 1.24 antibody having the antigen binding activity similar tothat of the chimeric anti-HM 1.24 antibody, it is necessary to convertamino acids included in FR3 or FR4 among those contained the V region ofthe H chain.

[0412] 2-4. Versions f to r of the H Chain

[0413] Version f of the H chain of the reshaped human anti-HM 1.24antibody was evaluated as mentioned for measurement of antigen bindingactivity. The result, as shown in FIG. 17, indicated that its antigenbinding activity is decreased as compared to the chimeric anti-HM 1.2.4antibody, but is increased as compared to the above versions a to c,suggesting that any of the four amino acids at position 67, 69, 75, and78 that were newly converted in this version is responsible for theactivity of the reshaped human antibody.

[0414] Version g of the H chain of the reshaped human anti-HM 1.24antibody was evaluated as mentioned for measurement of antigen bindingactivity. The result, as shown in FIG. 18 and 19, indicated that thisversion has exhibited a similar level of activity to that of the aboveversion a at most, revealing that, as shown for the above H chain humanmouse hybrid antibody, the amino acid at position 40 that was convertedin this version is not responsible for the increase in the activity ofthe reshaped human antibody.

[0415] Versions h to j of the H chain of the reshaped human anti-HM 1.24antibody were evaluated as mentioned for measurement of antigen bindingactivity and binding inhibition activity. The result, as shown in FIG.20, 21, 22, and 23, indicated that all versions were weaker for bothactivities as compared to the chimeric anti-HM 1.24 antibody and weresimilar to the above-mentioned version f, suggesting that the aminoacids at position 67 and 69 among the four amino acids that were newlyconverted in version f are not responsible for the increase in theactivity of the reshaped human antibody.

[0416] Versions k to p of the H chain of the reshaped human anti-HM 1.24antibody were evaluated as mentioned for measurement of antigen bindingactivity and binding inhibition activity. The result, as shown in FIG.24, 25, 26, and 27, indicated that all versions were weaker for bothactivities as compared to the chimeric anti-HM 1.24 antibody and weresimilar to the above-mentioned version h, suggesting that the aminoacids at position 80 and after that were newly converted in these sixversions are not responsible for the increase in the activity of thereshaped human antibody.

[0417] Version q of the H chain of the reshaped human anti-HM 1.24antibody was evaluated as mentioned for measurement of antigen bindingactivity and binding inhibition activity. The result, as shown in FIG.25 and 27, indicated that this version was weaker for both activities ascompared to the above version h or version p and was similar to that ofthe above-mentioned a, suggesting that substitution of the amino acid atposition 78 is essential for the increase in the activity of thereshaped human antibody.

[0418] Version r of the H chain of the reshaped human anti-HM 1.24antibody were evaluated by the method mentioned above. The result, asshown in FIG. 15 and 28, indicated that version r has a similar level ofantigen binding activity and binding inhibition activity to that of thechimeric anti-HM 1.24 antibody.

[0419] The above results indicated that the minimum conversion requiredfor the reshaped human anti-HM 1.24 antibody to have a similar level ofantigen binding activity to that of the mouse anti-HM 1.24 antibody orthe chimeric anti-HM 1.24 antibody is the amino acids at positions 30,71, and 78, and furthermore 73.

[0420] The antigen binding activity and the binding inhibition activityfor H chain versions a to r of the reshaped human anti-HM 1.24 antibodyare summarized in Table 6. TABLE 6 H chain version Antigen bindingactivity Binding inhibition activity a + + b + + c + + d + not measurede + not measured f ++ ++ g + + h ++ ++ i ++ ++ j ++ ++ k ++ ++ 1 ++ ++ m++ ++ n ++ ++ o ++ ++ p ++ ++ q + + r +++ +++

[0421] 2-5. Version s of the H Chain

[0422] Version s of the H chain of the reshaped human anti-HM 1.24antibody was evaluated in combination with the above-mentioned version aof the L chain as mentioned for measurement of antigen binding activityand binding inhibition activity. The result, as shown in FIG. 29 and 30,indicated that version s has a similar level of antigen binding activityand binding inhibition activity to that of version r.

[0423] As mentioned above, the reshaped human anti-HM 1.24 antibody ofthe present invention retains the ability of binding to antigen evenafter one or more amino acid residues have been replaced with otheramino acids. Accordingly, the present invention includes the reshapedhuman anti-HM 1.24 antibody in which one or more amino acid residueshave been replaced with other amino acids in the variable region of theH chain or the L chain as long as it retains the original properties.

[0424] 3. Evaluation of the Purified Reshaped Human Anti-HM 1.24Antibody

[0425] The purified reshaped human anti-HM 1.24 antibody was evaluatedfor the above-mentioned antigen binding activity and binding inhibitionactivity. The result, as shown in FIG. 31 and 32, indicated that thereshaped human anti-HM 1.24 antibody has a similar level of antigenbinding activity and binding inhibition activity to that of the chimericanti-HM 1.24 antibody. This fact indicated that the reshaped humananti-HM 1.24 antibody has the same antigen binding activity as the mouseanti-HM 1.24 antibody.

Example 12 Anti-Tumor Effect of the Chimeric Anti-HM 1.24 AntibodyAgainst the Human Myeloma Mouse Model

[0426] 1. Preparation of Antibody to be Administered

[0427] 1-1. Preparation of the Chimeric Anti-HM 1.24 Antibody

[0428] The purified chimeric anti-HM 1.24 antibody obtained in the aboveExample 6 was concentrated and the buffer solution was replaced byPBS(−) using the centrifuging ultrafiltration concentrator Centriprep 10(manufactured by Amicon). This was filter-sterilized using the membranefilter MILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22μm. This was prepared to a concentration of 200 μg/ml using thefilter-sterilized PBS(−), which was used for the following experiments.The concentration of the antibody was measured by absorbance at 280 nmand calculated with 1 mg/ml as 1.35 OD.

[0429] 1-2. Purification of the Control Human IgG1

[0430] Human IgG1 to be used as a control for the chimeric anti-HM 1.24antibody was purified as follows. After an equal amount of PBS(−) wasadded to Hu IgG1 Kappa Purified (manufactured by BINDING SITE), it wasaffinity-purified using the high-speed antibody purification systemConSep LC100 (manufactured by MILLIPORE) and Hyper D Protein A column(manufactured by Nippon Gaishi) using PBS(−) as the absorption bufferand 0.1 M sodium citrate buffer (pH 3) as the elution buffer accordingto the attached instructions. The eluted fractions were adjusted toabout pH 7.4 by immediately adding 1 M Tris-HCl (pH 8.0) and then usingthe centrifuging ultrafiltration concentrator Centriprep 10(manufactured by Amicon) concentration and buffer substitution to PBS(−)was carried out, and filter-sterilized using the membrane filterMILLEX-GV (manufactured by MILLIPORE) with a pore size of 0.22 μm. Thiswas adjusted to 200 μg/ml using the filter-sterilized PBS(−) and usedfor the following experiments. Antibody concentration was measured byabsorbance at 280 nm and calculated with 1 mg/ml as 1.35 OD.

[0431] 2. Method for Quantitating of Human Serum IgG in the Mouse Serum

[0432] Human IgG contained in the mouse serum was quantitated by thefollowing ELISA. 100 μl of goat anti-human IgG diluted to 1 μg/ml with0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate(manufactured by NUNC) and incubated at 4° C. overnight to immobilizethe antibody. After blocking, 100 μl of serially diluted mouse serum orhuman IgG as standard (manufactured by CAPPEL) was added and incubatedat room temperature for one hour. After washing, 100 μl of 2000-folddiluted alkaline phosphatase-labelled anti-human IgG (manufactured byCAPPEL) was added and incubated at room temperature for one hour. Afterwashing, the substrate solution was added and incubated, and thenabsorbance at 405 nm was measured using the MICROPLATE READER Model 3550(manufactured by Bio-Rad).

[0433] 3. Anti-Tumor Effect of the Chimeric Anti-HM 1.24 AntibodyAgainst the Human Myeloma Cells-Transplanted Mouse

[0434] 3-1. Construction the Human Myeloma Cells-Transplanted Mouse

[0435] The human myeloma cells-transplanted mouse was constructed asfollows. KPMM2 cells passaged in vivo using SCID mice (breeded by NihonCLEA) were prepared at a concentration of 3×10⁷ cells/ml with RPMI 1640medium supplemented with 10% fetal bovine serum (manufactured byGIBCOBRL). Two hundred μl of the above KPMM2 cell suspension wasinjected via the tail vein to SCID mice (male, 8-weeks old breeded byNihon CLEA) to which 100 μl of anti-asialo GM1 (manufactured by WakoPure Chemical Industries Co., Ltd.) had been intraperitoneally given onthe previous day.

[0436] 3-2. Administration of Antibody

[0437] On day 12 after KPMM2 cell transplantation, serum was collectedfrom the above human myeloma cells-transplanted mice, and human IgG inthe serum was quantitated using the ELISA mentioned in the above 2. Takeof KPMM2 cells in the bone marrow was confirmed by the increase of humanIgG level in the serum. On day 14, 21, and 28 after KPMM2 celltransplantation, 100 μl each of the antibodies prepared in the above 1was intraperitoneally given to these mice.

[0438] 3-3. Evaluation of the Anti-Tumor Effect of the Chimeric Anti-HM1.24 Antibody Against the Human Myeloma Cells-Transplanted Mouse

[0439] The anti-tumor effect of the chimeric anti-HM 1.24 antibody wasevaluated by the survival period of the mice. As shown in FIG. 33, themice that were given the chimeric anti-HM 1.24 antibody showed aprolonged period of survival as compared to the mice that receivedcontrol human IgG1. Thus, it was confirmed that the chimeric anti-HM1.24 antibody has the anti-tumor effect against the human myelomacells-transplanted mouse.

Example 13 Measurement of ADCC Activity of the Reshaped Human Anti-HM1.24 Antibody

[0440] ADCC (Antibody-dependent Cellular Cytotoxicity) activity wasmeasured according to the method as set forth in Current Protocols inImmunology, Chapter 7, Immunologic studies in humans, Editor, John E,Coligan et al., John Wiley & Sons, Inc., 1993.

[0441]1. Preparation of Effector Cells

[0442] Mononuclear cells were separated from the peripheral blood ofhealthy humans by the density centrifugation method. Thus, an equalamount of PBS(−) was added to the peripheral blood of healthy humans,which was layered on Ficoll-Paque PLUS (manufactured by Pharmacia), andwas centrifuged at 400 g for 40 minutes. The mononuclear cells layer wascollected, and washed four times with RPMI 1640 medium (manufactured byGIBCO BRL) supplemented with 10% fetal bovine serum (manufactured byGIBCO BRL), and prepared at a cell density of 5×10⁶/ml with the sameculture medium.

[0443] LAK (Limphokine Activated Killer Cell) was induced from the bonemarrow cells of SCID mice (breeded by Nihon CLEA). Thus, bone marrowcells were isolated from the femoral bone of the mice and washed twicewith RPMI1640 medium (manufactured by GIBCO BRL) supplemented with 10%fetal bovine serum (manufactured by GIBCO BRL), and prepared at a celldensity of 2×10⁵/ml with the same culture medium. This was incubatedtogether with 50 ng/ml of recombinant human IL-2 (manufactured by R & DSYSTEMS) and 10 ng/ml of recombinant mouse GM-CSF (manufactured by R & DSYSTEMS) in the CO₂ incubator (manufactured by TABAI) for seven days.The cell number was adjusted to 2×10⁶/ml with the same culture medium.

[0444] 2. Preparation of Target Cells

[0445] The human myeloma cell line KPMM2 (Japanese Unexamined PatentPublication (Kokai) No. 7-236475) or plasma cell leukemia-derived ARH-77(ATCC CCL-1621) was radiolabelled by incubating in the RPMI 1640 medium(manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum(manufactured by GIBCO BRL) together with 0.1 mCi of 51Cr-sodiumchromate (manufactured by ICN) at 37° C. for 60 minutes. Afterradiolabelling, the cells were washed three times with the same culturemedium and adjusted to 2×10⁵/ml.

[0446] 3. ADCC Assay

[0447] Into a 96-well U-bottomed plate (manufactured by BectonDickinson) were added 50 μl of 2×10⁵ target cells/ml, 50 μl of thereshaped human anti-HM 1.24 antibody, the mouse anti-HM 1.24 antibody,control human IgG1 (manufactured by THE BINDING SITE) or control mouseIgG2a (UPC10, manufactured by CAPPEL), and reacted at 4° C. for 15minutes.

[0448] Then, 100 μl of the effector cells was cultured in the CO₂incubator for 4 hours, when the ratio (E:T) of the effector cells (E) tothe target cells (T) was set at 0:1, 3.2:1, 8:1, 20:1, or 50:1.

[0449] One hundred μl of the supernatant was taken and the radioactivityreleased into the culture supernatant was measured by the gamma counter(ARC-300, manufactured by Aloka). For measurement of the maximumradioactivity, 1% NP-40 (manufactured by Nakalai) was used. Cytotoxicity(%) was calculated by (A−C)/(B−C)×100, wherein A is radioactivity (cpm)released in the presence of antibody, B is radioactivity (cpm) releasedby NP-40, and C is radioactivity (cpm) released by the culture mediumalone without antibody.

[0450]FIG. 34 shows the result obtained when the cells prepared from theperipheral blood from the healthy human were used as the effector celland KPMM2 cells were used as the target cell. FIG. 35 shows the resultobtained when the cells prepared from the peripheral blood from thehealthy human were used as the effector cell and ARH-77 was used as thetarget cell. When the reshaped human anti-HM 1.24 antibody was added,cytotoxicity increased with the increase in antibody concentration ascompared to the control human IgG1, indicating that the reshaped humananti-HM 1.24 antibody has ADCC activity.

[0451] Furthermore, when the reshaped human anti-HM 1.24 antibody wasadded, cytotoxicity evidently increased as compared to the mouse anti-HM1.24 antibody, indicating that the reshaped human anti-HM 1.24 antibodyhas higher ADCC activity than the mouse anti-HM 1.24 antibody.Furthermore, when KPMM2 was used as the target cell, the addition of thereshaped human anti-HM 1.24 antibody at a concentration of 0.1 μg/ml orhigher caused no change in cytotoxicity, indicating that theconcentration of 0.1 μg/ml or higher has sufficient ADCC activity. WhenARH-77 was used as the target cell, the addition of the reshaped humananti-HM 1.24 antibody at a concentration of 1 μg/ml or higher caused nochange in cytotoxicity, indicating that the concentration of 1 μg/ml orhigher has sufficient ADCC activity.

[0452]FIG. 36 shows the result obtained when the cells prepared from thebone marrow of SCID mice were used as the effector cell. When thereshaped human anti-HM 1.24 antibody was added, cytotoxicity increasedwith the increase in antibody concentration as compared to the controlhuman IgG1, indicating that the reshaped human anti-HM 1.24 antibody hasADCC activity. Furthermore, the addition of the reshaped human anti-HM1.24 antibody at a concentration of 0.1 μg/ml or higher caused no changein cytotoxicity, indicating that the concentration of 0.1 μg/ml orhigher has sufficient ADCC activity.

[0453] These results show that the reshaped human anti-HM 1.24 antibodyhas ADCC activity even when the effector cells used are derived fromhumans or mice.

Example 14 Anti-Tumor Effect of the Reshaped Anti-HM 1.24 AntibodyAgainst the Human Myeloma Mouse Model

[0454] 1. Preparation of Antibody to be Administered

[0455] The reshaped anti-HM 1.24 antibody obtained by introduction ofplasmid HEF-RVLa-AHM-gk and plasmid HEF-RVHr-AHM-gγ1 into CHO cells wasprepared to a concentration of 40, 200, and 1000 μg/ml using thefilter-sterilized PBS(−), and the control human IgG1 obtained in Example12.1-2 was prepared to a concentration of 200 μg/ml using thefilter-sterilized PBS(−), which were used as the antibodies to beadministered.

[0456] 2. Anti-Tumor Effect of the Reshaped Anti-HM 1.24 AntibodyAgainst the Human Myeloma Cells-Transplanted Mouse

[0457] 2-1. Construction of the Human Myeloma Cells-Transplanted Mouse

[0458] The human myeloma cells-transplanted mice were prepared accordingto Example 12.3-1. The mice used were SCID mice (five weeks old)(breeded by Nihon CLEA).

[0459] 2-2. The Administration of Antibodies

[0460] On day 9 after KPMM2 cell transplantation, serum was collectedfrom the above human myeloma cells-transplanted mice prepared in theabove 2-1, and human IgG in the serum was quantitated using the ELISAmentioned in the above 12.2. Take of KPMM2 cells on the bone marrow wasconfirmed by the increase of human IgG level in the serum. On day 10after KPMM2 cell transplantation, 100 μl each of the antibodies preparedin the above 1 was intravenously given to these mice.

[0461] 2-3. Evaluation of the Anti-Tumor Effect of the Reshaped Anti-HM1.24 Antibody Against the Human Myeloma Cells-Transplanted Mouse

[0462] The anti-tumor effect of the reshaped anti-HM 1.24 antibody wasevaluated by the change in the amount of human IgG in the mouse serumand in the survival period of mice.

[0463] The change in the amount of human IgG in the mouse serum wasquantitated for the serum collected on day 35 after the transplantationof KPMM2 cells by determining human IgG using the ELISA mentioned inExample 12.2. The result as shown in FIG. 37 revealed that in thecontrol human IgG1-administration group the amount of human IgG in theserum on day 35 after the KPMM2 cell transplantation was increased byabout 1000-fold as compared to that on day 9 (the day before antibodyadministration), whereas in the reshaped human anti-HM 1.24antibody-administration group it was almost equal to or below that onday 9 for any dosage, indicating that the reshaped human anti-HM 1.24antibody suppressed the growth of KPMM2 cells. On the other hand, forthe survival period as shown in FIG. 38, prolongation was observed forthe reshaped human anti-HM 1.24 antibody-administration group ascompared to the control human IgG1-administration group. The foregoingshows that the reshaped human anti-HM 1.24 antibody has the anti-tumoreffect against the human myeloma cells-transplanted mouse.

Example 15 Comparison of Anti-Tumor Effect Between the Reshaped HumanAnti-HM 1.24 Antibody and the Existing Drug Melphalan Against the HumanMyeloma Mouse Model

[0464] 1. Preparation of the Drugs to be Administered

[0465] 1-1. Preparation of Antibodies to be Administered

[0466] The reshaped human anti-HM 1.24 antibody obtained by theintroduction of plasmid HEF-RVLa-AHM-gk and plasmid HEF-RVHr-AHM-gγ1into CHO cells was prepared to a concentration of 40 and 200 μg/ml usingthe filter-sterilized PBS(−), and the control human IgG1 obtained inExample 12.1-2 was prepared to a concentration of 200 μg/ml using thefilter-sterilized PBS(−), which were used as the antibodies to beadministered.

[0467] 1-2. Preparation of Melphalan

[0468] Melphalan (manufactured by SIGMA) that is an existing drug formyeloma was prepared to a concentration of 0.1 mg/ml using 0.2%carboxymethyl cellulose (CMC) (manufactured by Daicel ChemicalIndustries, Ltd.).

[0469] 2. The Anti-Tumor Effect of the Reshaped Human Anti-HM 1.24Antibody and Melphalan Against the Human Myeloma Cells-TransplantedMouse

[0470] 2-1. Construction of Human Myeloma Cells-Transplanted Mouse

[0471] The human myeloma cells-transplanted mice were prepared accordingto Example 14.2-1.

[0472]2-2. The Administration of Drug

[0473] On day 9 after KPMM2 cells transplantation, serum was collectedfrom the above human myeloma cells-transplanted mice prepared in theabove 2-1, and human IgG in the serum was quantitated using the ELISAmentioned in the above 12.2. Take of KPMM2 cells on the bone marrow wasconfirmed by the increase of human IgG level in the serum. On day 10after KPMM2 cell transplantation, 100 μl each of the antibodies preparedin the above 1-1 were intravenously given to these mice. Furthermore,200 μl of 0.2% CMC solution was orally given once daily for five daysfrom day 10 after transplantation. On the other hand, for themelphalan-administration group, the melphalan solution prepared in theabove 1-2 was orally given at an amount of 100 μl per 10 g of bodyweight (1 mg/kg as melphalan) once daily for five days from day 10 aftertransplantation of KPMM2 cells.

[0474] 2-3. Evaluation of the Anti-Tumor Effect of the Reshaped Anti-HM1.24 Antibody Against the Human Myeloma Cells-Transplanted Mouse

[0475] The anti-tumor effect of the reshaped anti-HM 1.24 antibody wasevaluated by the change in the amount of human IgG in the mice serum andin the survival period of mice.

[0476] The change in the amount of human IgG in the mice serum wasquantitated for the serum collected on day 35 after the transplantationof KPMM2 cells by determining human IgG using the ELISA mentioned inExample 12.2. The result as shown in FIG. 39 revealed that in thecontrol human IgG1-administration group the amount of human IgG in theserum on day 35 after the KPMM2 cell transplantation was increased byabout 1000-fold as compared to that on day 9 (the day before antibodyadministration), whereas it seemed that KPMM2 cells grew in these mice.In the melphalan-administration group as well, the amount of serum humanIgG was more increased than that before the drug administration, thoughnot so high as in the control human IgG-administration group. Thisresult indicates that administration of melphalan did not suppress thegrowth of KPMM2 cells perfectly. On the other hand, in the reshapedhuman anti-HM 1.24 antibody-administration group, the amount of serumhuman IgG at day was less than at day 9 after transplantation for anydosage, indicating that the reshaped human anti-HM 1.24 antibodysuppressed the growth of KPMM2 cells.

[0477] On the other hand, for the survival period also as shown in FIG.40, prolongation was observed for the reshaped human anti-HM 1.24antibody-administration group as compared to the control humanIgG1-administration group or melphalan-administration group. From theforegoing, it was shown that the reshaped human anti-HM 1.24 antibodyhas the anti-tumor effect against the human myeloma cells-transplantedmice and that the anti-tumor effect of the present antibody is strongerthan the existing drug melphalan.

[0478] The above results indicated that when the human-derived effectorcells were used, the mouse anti-HM 1.24 antibody had little cytotoxicityto human myeloma cells, whereas the reshaped human anti-HM 1.24 antibodyand the chimeric anti-HM 1.24 antibody had strong cytotoxicity. Thisfact indicates the importance of humanizing antibody and provides hopeon the usefulness of the reshaped human anti-HM 1.24 antibody in humans.

[0479] The reshaped human anti-HM 1.24 antibody have exhibited a verystrong anti-tumor effect in the human myeloma cells-transplanted SCIDmice. Since in humans the effector cells are derived from humans andlymphocytes are normally present, an even stronger anti-tumor effect ofthe reshaped human anti-HM 1.24 antibody is expected.

[0480] In the myeloma model, the reshaped human anti-HM 1.24 antibodyhave exhibited a strong anti-tumor effect as compared to the existingdrug, and therefore, it is expected that the reshaped human anti-HM 1.24antibody will make an epoch-making drug for treatment of myeloma.

Reference Example 1 Construction of the Hybridoma that Produces theMouse Anti-HM 1.24 Monoclonal Antibody

[0481] The hybridoma that produces the mouse anti-HM 1.24 monoclonalantibody was prepared according to the method described in Goto, T. etal., Blood (1994) 84, 1992-1930.

[0482] The Epstein-Barr virus nuclear antigen (EBNA)-negative plasmacell line KPC-32 (1×10⁷ cells) derived from the bone marrow of humanpatient with multiple myeloma (Goto, T. et al., Jpn. J. Clin. Hematol.(11991) 32, 1400) was intraperitoneally given twice to BALB/c mice(breeded by Charles River) every six weeks.

[0483] In order to further elevate the titer of antibody production,1.5×10⁶ KPC-32 cells were injected into the spleen of the mise threedays before sacrificing the animals (Goto, T. et al., Tokushima J. Exp.Med. (1990) 37, 89). After sacrificing the mice, the spleen wereremoved, and the spleen cells removed according to the method of Groth,de St. & Schreidegger (Cancer Research (1981) 41, 3465) were subjectedto cell fusion with the myeloma cells SP2/0.

[0484] Antibody in the supernatant of the hybridoma culture was screenedby the ELISA (Posner, M.R. et al., J. Immunol. Methods (1982) 48, 23)using the KPC-32 cell-coated plates. 5×10⁴ KPC-32 cells were suspendedin 50 ml of PBS and aliquoted into 96-well plates (U-bottomed, Corning,manufactured by Iwaki). After blocking with PBS containing 1% bovineserum albumin (BSA), the supernatant of the hybridoma was added andincubated at 4° C. for 2 hours. Subsequently, it reacted withperoxidase-labelled goat anti-mouse IgG antibody (manufactured by Zymed)at 4° C. for 1 hour, washed once, and was reacted witho-phenylenediamine substrate solution (manufactured by SumitomoBakelite) at room temperature for 30 minutes.

[0485] After stopping the reaction with 2N sulfuric acid, absorbance at492 nm was measured using the ELISA reader (manufactured by Bio-Rad). Inorder to remove the hybridoma that produces antibody against humanimmunoglobulin, the positive hybridoma culture supernatant hadpreviously been adsorbed to human serum, and the reactivity to othersub-cellular components were screened. Positive hybridomas were selectedand their reactivity to various cell lines and human samples wereinvestigated using flow cytometry. The finally selected hybridoma cloneswere cloned twice, which were injected into the abdominal cavity of thepristane-treated BALB/c mice and then the ascitic fluid was obtainedtherefrom.

[0486] Monoclonal antibody was purified from the mouse ascites byammonium sulfate precipitation and Protein A affinity chromatography kit(Ampure Pa., manufactured by Amersham). The purified antibody wasconjugated to fluorescein isothiocyanate (FITC) using the Quick Tag FITCconjugation kit (manufactured by Boehringer Mannheim).

[0487] As a result, the monoclonal antibodies produced by 30 hybridomaclones reacted with KPC-32 and RPMI 8226 cells. After cloning, thereactivity of the supernatant of these hybridomas with other cell linesand peripheral blood-derived mononuclear cells was investigated.

[0488] Among them, three clones produced monoclonal antibodies thatspecifically react with plasma cells. Out of these three clones, thehybridoma clone that produce monocloned antibody that is most useful forflow cytometry analysis and that has complement-dependent cytotoxicityagainst RPUI 8226 cells was selected and termed HM1.24. The subclass ofmonoclonal antibody produced by this hybridoma was determined by theELISA using subclass-specific rabbit anti-mouse antibody (manufacturedby Zymed). Anti-HM 1.24 antibody had a subclass of IgG2a k. Thehybridoma that produces the anti-HM 1.24 antibody was internationallydeposited on Sep. 14, 1995, with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under theaccession number FERM BP-5233 under the provisions of the BudapestTreaty.

Reference Example 2 Cloning of cDNA Encoding the HM 1.24 AntigenPolypeptide

[0489] 1. Construction of cDNA Library

[0490] 1) Preparation of Total RNA

[0491] The cDNA that encodes the HM 1.24 antigen which is a polypeptidespecifically recognized by mouse anti-HM1.24 monoclonal antibody wasisolated as follows.

[0492] From the human multiple myeloma cell line KPMM2, total RNA wasprepared according to the method of Chirgwin et al. (Biochemistry, 18,5294 (1979)). Thus, 2.2×10⁸ KPMM2 cells were completely homogenized in20 ml of 4 M guanidine thiocyanate (manufactured by Nakalai tesque).

[0493] The homogenate was layered on 5.3 M cesium chloride layer in thecentrifuge tube, which was then centrifuged using Beckman SW40 rotor at31,000 rpm at 20° C. for 24 hours to precipitate RNA. The RNAprecipitate was washed with 70% ethanol, and dissolved in 300 μl of 10mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.5% SDS. After addingPronase (manufactured by Boehringer) thereto to a concentration of 0.5mg/ml, it was incubated at 37° C. for 30 minutes. The mixture wasextracted with phenol and chloroform to precipitate RNA. Then, the RNAprecipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.4)containing 1 mM EDTA.

[0494] 2) Preparation of Poly(A)+RNA

[0495] Using about 500 μg of the total RNA prepared as above as a rawmaterial, poly(A)+RNA was purified using the Fast Track 2.0 m RNAIsolation Kit (manufactured by Invitrogen) according to the instructionsattached to the kit.

[0496] 3) Construction of cDNA Library

[0497] Using 10 μg of the above poly(A)+RNA as a raw material, doublestranded cDNA was synthesized using the cDNA synthesizing kit TimeSavercDNA Synthesis Kit (manufactured by Pharmacia) according to theinstructions attached to the kit, and using the Directional CloningToolbox (manufactured by Pharmacia) EcoRI adapter was linked theretoaccording to the instructions attached to the kit. Kination andrestriction enzyme NotI treatment of the EcoRI adapter were carried outaccording to the instructions attached to the kit. Furthermore, theadapter-attached double strand cDNA having a size of about 500 bp orhigher was isolated and purified using 1.5% low melting point agarosegel (manufactured by SIGMA) to obtain about 40 μl of adapter-attacheddouble strand cDNA.

[0498] The adapter-attached double strand cDNA thus prepared was linkedto pCOS1 vector (Japanese Unexamined Patent Publication (Kokai)8-255196) that had previously been treated with restriction enzymesEcoRI and NotI and alkaline phosphatase (manufactured by Takara Shuzo)using T4 to construct DNA ligase (manufactured by GIBCO BRL) toconstruct cDNA library. The constructed cDNA library was transduced intoEscherichia coli strain DH5α (manufactured by GIBCO BRL) and the totalsize was estimated to be about 2.5×10⁶ independent clones.

[0499] 2. Cloning by Direct Expression

[0500] 1) Transfection into COS-7 Cells

[0501] cDNA was amplified by culturing about 5×10⁵ clones of the abovetransduced Escherichia coli in the 2-YT medium (Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press,(1989)) containing 50 μg/ml of ampicillin, and plasmid DNA was recoveredfrom the Escherichia coli by the alkali method (Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press,(1989)). The plasmid DNA obtained was transfected into COS-7 cells byelectroporation using the Gene Pulser instrument (manufactured byBioRad).

[0502] Thus, 10 μg of the purified plasmid DNA was added to 0.8 ml ofCOS-7 cells that were suspended into PBS at a concentration of 1×10⁷cells/ml, and was subjected to pulses at 1500 V and a capacity of 25 μF.After 10 minutes of recovery period at room temperature, theelectroporated cells were cultured in the DMEM (manufactured by GIBCOBRL) supplemented with 10% fetal bovine serum under the condition of 37°C. and 5% CO₂ for three days.

[0503] 2) Preparation of the Panning Dish

[0504] A panning dish coated with the mouse anti-HM 1.24 antibody wasprepared by the method of B. Seed et al. (Proc. Natl. Acad. Sci. USA,84, 3365-3369 (1987)). Thus, the mouse anti-HM 1.24 antibody was addedto 50 mM Tris-HCl, pH 9.5, to a concentration of 10 μg/ml. Three ml ofthe antibody solution thus prepared was added to a tissue culture platewith a diameter of 60 mm and incubated at room temperature for 2 hours.After washing three times with 0.15 M NaCl solution and blocking withPBS containing 5% fetal bovine serum, 1 mM EDTA, and 0.02% NaN₃, theseplates used for the following cloning.

[0505] 3) Cloning of cDNA Library

[0506] The COS-7 cells transfected as described above were detached byPBS containing 5 mM EDTA, and then washed once with PBS containing 5%fetal bovine serum. These cells were then suspended in PBS containing 5%fetal bovine serum and 0.02% NaN₃ to a concentration of about 1×10⁵cells/ml, which was added to the panning dish prepared as above andincubated at room temperature for 2 hours. After washing three timesgently with PBS containing 5% fetal bovine serum and 0.02% NaN₃, plasmidDNA was recovered from the cells bound to the panning dish using asolution containing 0.6% SDS and 10 mM EDTA.

[0507] The recovered plasmid DNA was transduced again to Escherichiacoli DH5α. After amplifying plasmid DNA as above, it was recovered bythe alkali method. The recovered plasmid DNA was transfected into COS-7cells by the electroporation method and plasmid DNA recovered from thebound cells as described above. The same procedure was repeated one moretime, and the recovered plasmid DNA was digested with restrictionenzymes EcoRI and NotI. As a result, concentration of the insert with asize of about 0.9 kbp was confirmed. Escherichia coli transduced withpart of the recovered plasmid DNA was inoculated to the 2-YT agar platecontaining 50 μg/ml of ampicillin. After culturing overnight, plasmidDNA was recovered from single colony. It was digested with restrictionenzymes EcoRI and NotI and clone p3.19 having an insert of 0.9 kbp wasobtained.

[0508] The base sequence of this clone was determined by reacting usingPRISM, Dye Terminater Cycle Sequencing kit (manufactured by PerkinElmer) according to the instructions attached to the kit and sequencingusing ABI 373A DNA Sequencer (manufactured by Perkin Elmer). The aminoacid sequence and the base sequence thereof are shown in SEQ ID NO: 103.

[0509] The cDNA encoding the polypeptide having the amino acid sequenceas set forth in SEQ ID NO: 103 was inserted into the XbaI cleavage siteof pUC19 vector, and has been prepared as plasmid pRS38-pUC19. TheEscherichia coli that contains this plasmid pRS38-pUC19 has beeninternationally deposited on Oct. 5,1993, as Escherichia coli DH5α(pRS38-pUC19), with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture, Japan) under theaccession number FERM BP-4434 under the provisions of the BudapestTreaty (see Japanese Unexamined Patent Publication (Kokai) No.7-196694).

[0510] Industrial Applicability

[0511] Since the chimeric anti-HM 1.24 antibody is composed of thevariable region of the mouse anti-HM 1.24 antibody and the constantregion of a human antibody, and the reshaped human anti-HM 1.24 antibodyis composed of the complementarity determining region of the mouseanti-HM 1.24 antibody, the framework region of a human antibody, and theconstant region of a human antibody, it has a low antigenicity againsthumans, and therefore, is expected to be used as a medical composition,especially for treatment of myeloma.

REFERENCE TO THE ORGANISMS DONATED

[0512] The international depository concerned

[0513] Title: the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, MITI

[0514] Address: Higashi 1-Chome 1-3, Tsukuba city, Ibalaki prefecture,Japan

[0515] 1. Escherichia coli DH5α (pRS 38-pUC19)

[0516] Accession No.: FERM BP-4434

[0517] Date of donation: Oct. 5, 1993

[0518]2. Mouse-mouse hybridoma HM1.24

[0519] Accession No.: FERM BP-5233

[0520] Date of donation: Apr. 27, 1995

[0521]3. Escherichia coli DH5α (pUC19-RVHr-AHM-gγ1)

[0522] Accession No.: FERM BP-5643

[0523] Date of donation: Aug. 29, 1996

[0524]4. Escherichia coli DH5α (pUC19-1.24H-gγ1)

[0525] Accession No.: FERM BP-5644

[0526] Date of donation: Aug. 29, 1996

[0527]5. Escherichia coli DH5α (pUC19-RVLa-AHM-gk)

[0528] Accession No.: FERM BP-5645

[0529] Date of donation: Aug. 29, 1996

[0530]6. Escherichia coli DH5α (pUC19-1.24L-gk)

[0531] Accession No.: FERM BP-5646

[0532] Date of donation: Aug. 29, 1996

[0533]7. Escherichia coli DH5α (pUC19-RVHs-AHM-gγ1)

[0534] Accession No.: FERM BP-6127

[0535] Date of donation: Sep. 29, 1997

1 137 1 394 DNA murine CDS (1)..(393) sig_peptide (1)..(72) mat_peptide(73)..(393) Nucleotide sequence coding for L chain V region of mouseanti-HM 1.24 antibody 1 atg ggc ttc aag atg gag tca cat ttt ctg gtc tttgta ttc gtg ttt 48 Met Gly Phe Lys Met Glu Ser His Phe Leu Val Phe ValPhe Val Phe -20 -15 -10 ctc tgg ttg tct ggt gtt gac gga gac att gtg atgacc cag tct cac 96 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met ThrGln Ser His -5 -1 1 5 aaa ttc atg tcc aca tca gta gga gac agg gtc agcatc acc tgc aag 144 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser IleThr Cys Lys 10 15 20 gcc agt cag gat gtg aat act gct gta gcc tgg tat caacaa aaa cca 192 Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln GlnLys Pro 25 30 35 40 gga caa tcg cct aaa cta ctg att tac tcg gca tcc aaccgg tac act 240 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn ArgTyr Thr 45 50 55 gga gtc cct gat cgc atc act ggc agt gga tct ggg acg gatttc act 288 Gly Val Pro Asp Arg Ile Thr Gly Ser Gly Ser Gly Thr Asp PheThr 60 65 70 ttc acc atc agc agt gtg cag gcg gaa gac ctg gca ctt tat tactgt 336 Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys75 80 85 cag caa cat tat agt act cca ttc acg ttc ggc tcg ggg aca aag ttg384 Gln Gln His Tyr Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 9095 100 gaa ata aaa c 394 Glu Ile Lys 105 2 418 DNA murine CDS (1)..(417)sig_peptide (1)..(57) mat_peptide (58)..(417) Nucleotide sequence codingfor H chain V region of mouse anti-HM 1.24 antibody 2 atg gaa tgt aactgg ata ctt cct ttt att ctg tca gta act tca ggt 48 Met Glu Cys Asn TrpIle Leu Pro Phe Ile Leu Ser Val Thr Ser Gly -15 -10 -5 gcc tac tca caggtt caa ctc cag cag tct ggg gct gag ctg gca aga 96 Ala Tyr Ser Gln ValGln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg -1 1 5 10 cct ggg gct tcagtg aag ttg tcc tgc aag gct tct ggc tac acc ttt 144 Pro Gly Ala Ser ValLys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atgcag tgg gta aaa cag agg cct gga cag ggt ctg 192 Thr Pro Tyr Trp Met GlnTrp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 gaa tgg att ggg tctatt ttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Ile Gly Ser IlePhe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aaggcc aca ttg act gca gat aaa tcc tcc agt 288 Gln Lys Phe Lys Gly Lys AlaThr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 aca gcc tac atg caa ctc agcatc ttg gca ttt gag gac tct gcg gtc 336 Thr Ala Tyr Met Gln Leu Ser IleLeu Ala Phe Glu Asp Ser Ala Val 80 85 90 tat tac tgt gca aga gga tta cgacga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg ArgGly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggc caa ggc acc act ctc acagtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115120 3 11 PRT murine Amino acid sequence of L chain V region CDR(1) ofmouse anti-HM 1.24 antibody 3 Lys Ala Ser Gln Asp Val Asn Thr Ala ValAla 1 5 10 4 7 PRT murine Amino acid sequence of CDR(2) of L chain Vregion of mouse anti-HM 1.24 antibody 4 Ser Ala Ser Asn Arg Tyr Thr 1 55 9 PRT murine Amino acid sequence of CDR(3) of L chain V region ofmouse anti-HM 1.24 antibody 5 Gln Gln His Tyr Ser Thr Pro Phe Thr 1 5 65 PRT murine Amino acid sequence of CDR(1) of H chain V region of mouseanti-HM 1.24 antibody 6 Pro Tyr Trp Met Gln 1 5 7 17 PRT murine Aminoacid sequence of CDR(2) of H chain V region of mouse anti-HM 1.24antibody 7 Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys PheLys 1 5 10 15 Gly 8 11 PRT murine Amino acid sequence of CDR(3) of Hchain V region of mouse anti-HM 1.24 antibody 8 Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 1 5 10 9 379 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody L chain V region version a 9 atg gga tgg agc tgtatc atc ctc tcc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys IleIle Leu Ser Leu Val Ala Thr Ala Thr Gly -15 -10 -5 gtc cac tcc gac atccag atg acc cag agc cca agc agc ctg agc gcc 96 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala -1 1 5 10 agc gtg ggt gac agagtg acc atc acc tgt aag gct agt cag gat gtg 144 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp Val 15 20 25 aat act gct gta gcc tggtac cag cag aag cca gga aag gct cca aag 192 Asn Thr Ala Val Ala Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 ctg ctg atc tac tcg gcatcc aac cgg tac act ggt gtg cca agc aga 240 Leu Leu Ile Tyr Ser Ala SerAsn Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60 ttc agc ggt agc ggt agc ggtacc gac ttc acc ttc acc atc agc agc 288 Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Phe Thr Ile Ser Ser 65 70 75 ctc cag cca gag gac atc gct acctac tac tgc cag caa cat tat agt 336 Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln His Tyr Ser 80 85 90 act cca ttc acg ttc ggc caa ggg accaag gtg gaa atc aaa c 379 Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 95 100 105 10 379 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody L chain V region version b 10 atg gga tgg agc tgtatc atc ctc tcc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys IleIle Leu Ser Leu Val Ala Thr Ala Thr Gly -15 -10 -5 gtc cac tcc gac atccag atg acc cag agc cca agc agc ctg agc gcc 96 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala -1 1 5 10 agc gtg ggt gac agagtg acc atc acc tgt aag gct agt cag gat gtg 144 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asp Val 15 20 25 aat act gct gta gcc tggtac cag cag aag cca gga aag gct cca aag 192 Asn Thr Ala Val Ala Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 ctg ctg atc tac tcg gcatcc aac cgg tac act ggt gtg cca agc aga 240 Leu Leu Ile Tyr Ser Ala SerAsn Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60 ttc agc ggt agc ggt agt ggtacc gac tac acc ttc acc atc agc agc 288 Phe Ser Gly Ser Gly Ser Gly ThrAsp Tyr Thr Phe Thr Ile Ser Ser 65 70 75 ctc cag cca gag gac atc gct acctac tac tgc cag caa cat tat agt 336 Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln His Tyr Ser 80 85 90 act cca ttc acg ttc ggc caa ggg accaag gtg gaa atc aaa c 379 Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 95 100 105 11 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version a 11 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtcacc atg acc gca gac acg tcc acg agc 288 Gln Lys Phe Lys Gly Arg Val ThrMet Thr Ala Asp Thr Ser Thr Ser 65 70 75 aca gtc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 12418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version b 12 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtc acc atg acc gca gac acgtcc acg agc 288 Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr SerThr Ser 65 70 75 aca gtc tac atg gag ctg agc agc ctg aga tct gag gac acggcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 13 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version c 13atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aga gtc act atg acc gca gac aag tcc acg agc 288 Gln Lys PheLys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser 65 70 75 aca gtc tacatg gag ctg agc agc ctg aga tct gag gac acg gcc gtg 336 Thr Val Tyr MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 14 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version d 14 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtcacc atg acc gca gac aag tcc acg agc 288 Gln Lys Phe Lys Gly Lys Val ThrMet Thr Ala Asp Lys Ser Thr Ser 65 70 75 aca gtc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 15418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version e 15 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gcc acc ctg acc gca gac acgtcc acg agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr SerThr Ser 65 70 75 aca gtc tac atg gag ctg agc agc ctg aga tct gag gac acggcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 16 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version f 16atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aga gcc acc ctg act gca gac acg tcc tcg agc 288 Gln Lys PheLys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tacatg gag ctg agc agc ctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 17 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version g 17 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag cgc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtcacc atg acc gca gac acg tcc acg agc 288 Gln Lys Phe Lys Gly Arg Val ThrMet Thr Ala Asp Thr Ser Thr Ser 65 70 75 aca gtc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 18418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version h 18 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtc acc atg acc gca gac acgtcc tcg agc 288 Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr SerSer Ser 65 70 75 aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acggcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 19 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version i 19atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aaa gtc acc atg acc gca gac acg tcc tcg agc 288 Gln Lys PheLys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tacatg gag ctg agc agc ctg gca ttt gag gac acg gcc gtg 336 Thr Ala Tyr MetGlu Leu Ser Ser Leu Ala Phe Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 20 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version j 20 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gccacc ctg act gca gac acg tcc tcg agc 288 Gln Lys Phe Lys Gly Lys Ala ThrLeu Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 21418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version k 21 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtc acc atg acc gca gac acgtcc tcg agc 288 Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr SerSer Ser 65 70 75 aca gcc tac atg cag ctg agc agc cta aga tct gag gac acggcc gtg 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 22 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version l 22atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aaa gtc acc atg acc gca gac acg tcc tcg agc 288 Gln Lys PheLys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tacatg cag ctg agc atc ctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr MetGln Leu Ser Ile Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 23 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version m 23 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtcacc atg acc gca gac acg tcc tcg agc 288 Gln Lys Phe Lys Gly Lys Val ThrMet Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tac atg cag ctg agc atcctg aga tct gag gac tcg gcc gtg 336 Thr Ala Tyr Met Gln Leu Ser Ile LeuArg Ser Glu Asp Ser Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 24418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version n 24 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aaa gtc acc atg acc gca gac acgtcc tcg agc 288 Gln Lys Phe Lys Gly Lys Val Thr Met Thr Ala Asp Thr SerSer Ser 65 70 75 aca gcc tac atg gag ctg agc atc ctg aga tct gag gac acggcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ile Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 25 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version o 25atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aaa gtc acc atg acc gca gac acg tcc tcg agc 288 Gln Lys PheLys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 aca gcc tacatg gag ctg agc agc ctg aga tct gag gac tcg gcc gta 336 Thr Ala Tyr MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 26 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version p 26 atg gac tgg acc tggagg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 Met Asp Trp Thr Trp ArgVal Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 gct cac tcc cag gtgcag ctg gtg cag tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtgaag gtt tcc tgc aag gca tct gga tac acc ttc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cagtgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln TrpVal Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gag tgg atg gga tct attttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile PhePro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtcacc atg acc gca gac acg tcc acg agc 288 Gln Lys Phe Lys Gly Arg Val ThrMet Thr Ala Asp Thr Ser Thr Ser 65 70 75 aca gcc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcg aga gga tta cga cgaggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg GlyGly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtctcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 27418 DNA Artificial Sequence Description of Artificial SequenceNucleotide sequence coding for reshaped human anti-HM 1.24 antibody Hchain V region version q 27 atg gac tgg acc tgg agg gtc ttc ttc ttg ctggct gta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggggct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gcatct gga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cctgga caa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro GlyGln Gly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gatact agg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp ThrArg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtc acc atg acc gca gac acgtcc tcg agc 288 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr SerSer Ser 65 70 75 aca gtc tac atg gag ctg agc agc ctg aga tct gag gac acggcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gactac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 28 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region version r 28atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct cca ggt 48 MetAsp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag 96 AlaHis Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac acc ttc 144 ProGly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 actccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt 192 Thr ProTyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 gagtgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt 240 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aagttc aag ggc aga gtc acc atg acc gca gac aag tcc acg agc 288 Gln Lys PheLys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser 65 70 75 aca gcc tacatg gag ctg agc agc ctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr MetGlu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 tat tac tgt gcgaga gga tta cga cga ggg ggg tac tac ttt gac tac 384 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 tgg ggg caa gggacc acg gtc acc gtc tcc tca g 418 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 110 115 120 29 40 DNA Artificial Sequence Description ofArtificial Sequence primer 29 actagtcgac atgaagttgc ctgttaggctgttggtgctg 40 30 39 DNA Artificial Sequence Description of ArtificialSequence primer 30 actagtcgac atggagwcag acacactcct gytatgggt 39 31 40DNA Artificial Sequence Description of Artificial Sequence primer 31actagtcgac atgagtgtgc tcactcaggt cctggsgttg 40 32 43 DNA ArtificialSequence Description of Artificial Sequence primer 32 actagtcgacatgaggrccc ctgctcagwt tyttggmwtc ttg 43 33 40 DNA Artificial SequenceDescription of Artificial Sequence primer 33 actagtcgac atggatttwcaggtgcagat twtcagcttc 40 34 37 DNA Artificial Sequence Description ofArtificial Sequence primer 34 actagtcgac atgaggtkcy ytgytsagyt yctgrgg37 35 41 DNA Artificial Sequence Description of Artificial Sequenceprimer 35 actagtcgac atgggcwtca agatggagtc acakwyycwg g 41 36 41 DNAArtificial Sequence Description of Artificial Sequence primer 36actagtcgac atgtggggay ctktttycmm tttttcaatt g 41 37 35 DNA ArtificialSequence Description of Artificial Sequence primer 37 actagtcgacatggtrtccw casctcagtt ccttg 35 38 37 DNA Artificial Sequence Descriptionof Artificial Sequence primer 38 actagtcgac atgtatatat gtttgttgtctatttct 37 39 38 DNA Artificial Sequence Description of ArtificialSequence primer 39 actagtcgac atggaagccc cagctcagct tctcttcc 38 40 27DNA Artificial Sequence Description of Artificial Sequence primer 40ggatcccggg tggatggtgg gaagatg 27 41 25 DNA Artificial SequenceDescription of Artificial Sequence primer 41 tagagtcacc gaggagccag ttgta25 42 26 DNA Artificial Sequence Description of Artificial Sequenceprimer 42 ggatcccggg agtggataga ccgatg 26 43 34 DNA Artificial SequenceDescription of Artificial Sequence primer 43 gataagcttc caccatgggcttcaagatgg agtc 34 44 34 DNA Artificial Sequence Description ofArtificial Sequence primer 44 gataagcttc caccatggaa tgtaactgga tact 3445 34 DNA Artificial Sequence Description of Artificial Sequence primer45 ggcggatcca ctcacgtttt atttccaact ttgt 34 46 34 DNA ArtificialSequence Description of Artificial Sequence primer 46 ggcggatccactcacctgag gagactgtga gagt 34 47 18 DNA Artificial Sequence Descriptionof Artificial Sequence primer 47 cagacagtgg ttcaaagt 18 48 26 DNAArtificial Sequence Description of Artificial Sequence primer 48gaattcggat ccactcacgt ttgatt 26 49 48 DNA Artificial SequenceDescription of Artificial Sequence primer 49 agtcaggatg tgaatactgctgtagcctgg taccagcaga agccagga 48 50 39 DNA Artificial SequenceDescription of Artificial Sequence primer 50 gcatccaacc ggtacactggtgtgccaagc agattcagc 39 51 45 DNA Artificial Sequence Description ofArtificial Sequence primer 51 caacattata gtactccatt cacgttcggccaagggacca aggtg 45 52 47 DNA Artificial Sequence Description ofArtificial Sequence primer 52 gcagtattca catcctgact ggccttacaggtgatggtca ctctgtc 47 53 38 DNA Artificial Sequence Description ofArtificial Sequence primer 53 acaccagtgt accggttgga tgccgagtag atcagcag38 54 41 DNA Artificial Sequence Description of Artificial Sequenceprimer 54 gtgaatggag tactataatg ttgctggcag tagtaggtag c 41 55 31 DNAArtificial Sequence Description of Artificial Sequence primer 55ggtaccgact acaccttcac catcagcagc c 31 56 31 DNA Artificial SequenceDescription of Artificial Sequence primer 56 ggtgaaggtg tagtcggtaccgctaccgct a 31 57 144 DNA Artificial Sequence Description of ArtificialSequence DNA fragment for construction of H chain V region 57 atgccttgcaggaaaccttc actgaggccc caggcttctt cacctcagcc ccagactgca 60 ccagctgcacctgggagtga gcacctggag ctacagccag caagaagaag accctccagg 120 tccagtccatggtggaagct tatc 144 58 130 DNA Artificial Sequence Description ofArtificial Sequence DNA fragment for construction of H chain V region 58tcagtgaagg tttcctgcaa ggcatctgga tacaccttca ctccctactg gatgcagtgg 60gtgcgacagg cccctggaca agggcttgag tggatgggat ctatttttcc tggagatggt 120gatactaggt 130 59 131 DNA Artificial Sequence Description of ArtificialSequence DNA fragment for construction of H chain V region 59 aatacacggccgtgtcctca gatctcaggc tgctcagctc catgtagact gtgctcgtgg 60 acgtgtctgcggtcatggtg actctgccct tgaacttctg actgtaccta gtatcaccat 120 ctccaggaaa a131 60 119 DNA Artificial Sequence Description of Artificial SequenceDNA fragment for construction of H chain V region 60 gagatctgaggacacggccg tgtattactg tgcgagagga ttacgacgag gggggtacta 60 ctttgactactgggggcaag ggaccacggt caccgtctcc tcaggtgagt ggatccgac 119 61 25 DNAArtificial Sequence Description of Artificial Sequence primer 61gataagcttc caccatggac tggac 25 62 25 DNA Artificial Sequence Descriptionof Artificial Sequence primer 62 gtcggatcca ctcacctgag gagac 25 63 26DNA Artificial Sequence Description of Artificial Sequence primer 63aagttcaagg gcaaagtcac catgac 26 64 26 DNA Artificial SequenceDescription of Artificial Sequence primer 64 gtcatggtga ctttgcccttgaactt 26 65 26 DNA Artificial Sequence Description of ArtificialSequence primer 65 atgaccgcag acaagtccac gagcac 26 66 26 DNA ArtificialSequence Description of Artificial Sequence primer 66 gtgctcgtggacttgtctgc ggtcat 26 67 47 DNA Artificial Sequence Description ofArtificial Sequence primer 67 aagttcaagg gcaaagtcac catgaccgcagacaagtcca cgagcac 47 68 47 DNA Artificial Sequence Description ofArtificial Sequence primer 68 gtgctcgtgg acttgtctgc ggtcatggtgactttgccct tgaactt 47 69 38 DNA Artificial Sequence Description ofArtificial Sequence primer 69 aagttcaagg gcagagccac cctgaccgca gacacgtc38 70 38 DNA Artificial Sequence Description of Artificial Sequenceprimer 70 gacgtgtctg cggtcagggt ggctctgccc ttgaactt 38 71 18 DNAArtificial Sequence Description of Artificial Sequence primer 71cagacagtgg ttcaaagt 18 72 17 DNA Artificial Sequence Description ofArtificial Sequence primer 72 gccccaaagc caaggtc 17 73 23 DNA ArtificialSequence Description of Artificial Sequence primer 73 atttttcctggagatggtga tac 23 74 23 DNA Artificial Sequence Description ofArtificial Sequence primer 74 gtatcaccat ctccaggaaa tat 23 75 418 DNAArtificial Sequence Description of Artificial Sequence Nucleotidesequence coding for reshaped human anti-HM 1.24 antibody H chain Vregion (mouse-human hybrid) 75 atg gaa tgt aac tgg ata ctt cct ttt attctg tca gta act tca ggt 48 Met Glu Cys Asn Trp Ile Leu Pro Phe Ile LeuSer Val Thr Ser Gly -15 -10 -5 gcc tac tca cag gtt caa ctc cag cag tctggg gct gag ctg gca aga 96 Ala Tyr Ser Gln Val Gln Leu Gln Gln Ser GlyAla Glu Leu Ala Arg -1 1 5 10 cct ggg gct tca gtg aag ttg tcc tgc aaggct tct ggc tac acc ttt 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys AlaSer Gly Tyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gta aaa cag aggcct gga cag ggt ctg 192 Thr Pro Tyr Trp Met Gln Trp Val Lys Gln Arg ProGly Gln Gly Leu 30 35 40 45 gaa tgg att ggg tct att ttt cct gga gat ggtgat act agg tac agt 240 Glu Trp Ile Gly Ser Ile Phe Pro Gly Asp Gly AspThr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtc acc atg acc gca gacacg tcc acg agc 288 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp ThrSer Thr Ser 65 70 75 aca gtc tac atg gag ctg agc agc ctg aga tct gag gacacg gcc gtg 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Val 80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac tttgac tac 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe AspTyr 95 100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp GlyGln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 76 418 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor reshaped human anti-HM 1.24 antibody H chain V region (human-mousehybrid) 76 atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct ccaggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly-15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg aagaag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys-1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gca tct gga tac accttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cct gga caa ggg ctt192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 3035 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gat act agg tac agt240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 5055 60 cag aag ttc aag ggc aag gcc aca ttg act gca gat aaa tcc tcc agt288 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 6570 75 aca gcc tac atg caa ctc agc atc ttg gca ttt gag gac tct gcg gtc336 Thr Ala Tyr Met Gln Leu Ser Ile Leu Ala Phe Glu Asp Ser Ala Val 8085 90 tat tac tgt gca aga gga tta cga cga ggg ggg tac tac ttt gac tac384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95100 105 tgg ggc caa ggc acc act ctc aca gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Leu Thr Val Ser Ser 110 115 120 77 38 DNA ArtificialSequence Description of Artificial Sequence primer 77 ctggttcggcccacctctga aggttccaga atcgatag 38 78 35 DNA Artificial SequenceDescription of Artificial Sequence primer 78 gcagacacgt cctcgagcacagcctacatg gagct 35 79 35 DNA Artificial Sequence Description ofArtificial Sequence primer 79 agctccatgt aggctgtgct cgaggacgtg tctgc 3580 26 DNA Artificial Sequence Description of Artificial Sequence primer80 tgggtgcgac agcgccctgg acaagg 26 81 26 DNA Artificial SequenceDescription of Artificial Sequence primer 81 ccttgtccag ggcgctgtcgcaccca 26 82 41 DNA Artificial Sequence Description of ArtificialSequence primer 82 tacatggagc tgagcagcct ggcatttgag gacacggccg t 41 8341 DNA Artificial Sequence Description of Artificial Sequence primer 83acggccgtgt cctcaaatgc caggctgctc agctccatgt a 41 84 26 DNA ArtificialSequence Description of Artificial Sequence primer 84 aagttcaagggcaaagccac cctgac 26 85 26 DNA Artificial Sequence Description ofArtificial Sequence primer 85 gtcagggtgg ctttgccctt gaactt 26 86 23 DNAArtificial Sequence Description of Artificial Sequence primer 86gcctacatgc agctgagcag cct 23 87 23 DNA Artificial Sequence Descriptionof Artificial Sequence primer 87 aggctgctca gctgcatgta ggc 23 88 38 DNAArtificial Sequence Description of Artificial Sequence primer 88gcctacatgc agctgagcat cctgagatct gaggacac 38 89 35 DNA ArtificialSequence Description of Artificial Sequence primer 89 gatctcaggatgctcagctg catgtaggct gtgct 35 90 50 DNA Artificial Sequence Descriptionof Artificial Sequence primer 90 gcctacatgc agctgagcat cctgagatctgaggactcgg ccgtgtatta 50 91 50 DNA Artificial Sequence Description ofArtificial Sequence primer 91 acggccgagt cctcagatct caggatgctcagctgcatgt aggctgtgct 50 92 20 DNA Artificial Sequence Description ofArtificial Sequence primer 92 gagctgagca tcctgagatc 20 93 26 DNAArtificial Sequence Description of Artificial Sequence primer 93gatctcagga tgctcagctc catgta 26 94 20 DNA Artificial SequenceDescription of Artificial Sequence primer 94 agatctgagg actcggccgt 20 9520 DNA Artificial Sequence Description of Artificial Sequence primer 95acggccgagt cctcagatct 20 96 35 DNA Artificial Sequence Description ofArtificial Sequence primer 96 gcagacacgt ccacgagcac agcctacatg gagct 3597 35 DNA Artificial Sequence Description of Artificial Sequence primer97 agctccatgt aggctgtgct cgtggacgtg tctgc 35 98 35 DNA ArtificialSequence Description of Artificial Sequence primer 98 gcagacacgtcctcgagcac agtctacatg gagct 35 99 35 DNA Artificial Sequence Descriptionof Artificial Sequence primer 99 agctccatgt agactgtgct cgaggacgtg tctgc35 100 26 DNA Artificial Sequence Description of Artificial Sequenceprimer 100 agagtcacca tcaccgcaga caagtc 26 101 26 DNA ArtificialSequence Description of Artificial Sequence primer 101 gacttgtctgcggtgatggt gactct 26 102 418 DNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence coding for reshaped humananti-HM 1.24 antibody H chain V region version s inserted intoHEF-RVHs-AHM-g(y)l 102 atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gctgta gct cca ggt 48 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala ValAla Pro Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tct ggg gctgag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gca tctgga tac acc ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser GlyTyr Thr Phe 15 20 25 act ccc tac tgg atg cag tgg gtg cga cag gcc cct ggacaa ggg ctt 192 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala Pro Gly GlnGly Leu 30 35 40 45 gag tgg atg gga tct att ttt cct gga gat ggt gat actagg tac agt 240 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr ArgTyr Ser 50 55 60 cag aag ttc aag ggc aga gtc acc atc acc gca gac aag tccacg agc 288 Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser ThrSer 65 70 75 aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gccgtg 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val80 85 90 tat tac tgt gcg aga gga tta cga cga ggg ggg tac tac ttt gac tac384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95100 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly GlnGly Thr Thr Val Thr Val Ser Ser 110 115 120 103 1013 DNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence codingfor HM 1.24 antigen 103 gaattcggca cgagggatct gg atg gca tct act tcg tatgac tat tgc aga 52 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg 1 5 10 gtgccc atg gaa gac ggg gat aag cgc tgt aag ctt ctg ctg ggg ata 100 Val ProMet Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile 15 20 25 gga attctg gtg ctc ctg atc atc gtg att ctg ggg gtg ccc ttg att 148 Gly Ile LeuVal Leu Leu Ile Ile Val Ile Leu Gly Val Pro Leu Ile 30 35 40 atc ttc accatc aag gcc aac agc gag gcc tgc cgg gac ggc ctt cgg 196 Ile Phe Thr IleLys Ala Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg 45 50 55 gca gtg atg gagtgt cgc aat gtc acc cat ctc ctg caa caa gag ctg 244 Ala Val Met Glu CysArg Asn Val Thr His Leu Leu Gln Gln Glu Leu 60 65 70 acc gag gcc cag aagggc ttt cag gat gtg gag gcc cag gcc gcc acc 292 Thr Glu Ala Gln Lys GlyPhe Gln Asp Val Glu Ala Gln Ala Ala Thr 75 80 85 90 tgc aac cac act gtgatg gcc cta atg gct tcc ctg gat gca gag aag 340 Cys Asn His Thr Val MetAla Leu Met Ala Ser Leu Asp Ala Glu Lys 95 100 105 gcc caa gga caa aagaaa gtg gag gag ctt gag gga gag atc act aca 388 Ala Gln Gly Gln Lys LysVal Glu Glu Leu Glu Gly Glu Ile Thr Thr 110 115 120 tta aac cat aag cttcag gac gcg tct gca gag gtg gag cga ctg aga 436 Leu Asn His Lys Leu GlnAsp Ala Ser Ala Glu Val Glu Arg Leu Arg 125 130 135 aga gaa aac cag gtctta agc gtg aga atc gcg gac aag aag tac tac 484 Arg Glu Asn Gln Val LeuSer Val Arg Ile Ala Asp Lys Lys Tyr Tyr 140 145 150 ccc agc tcc cag gactcc agc tcc gct gcg gcg ccc cag ctg ctg att 532 Pro Ser Ser Gln Asp SerSer Ser Ala Ala Ala Pro Gln Leu Leu Ile 155 160 165 170 gtg ctg ctg ggcctc agc gct ctg ctg cag tgagatccca ggaagctggc 582 Val Leu Leu Gly LeuSer Ala Leu Leu Gln 175 180 acatcttgga aggtccgtcc tgctcggctt ttcgcttgaacattcccttg atctcatcag 642 ttctgagcgg gtcatggggc aacacggtta gcggggagagcacggggtag ccggagaagg 702 gcctctggag caggtctgga ggggccatgg ggcagtcctgggtctgggga cacagtcggg 762 ttgacccagg gctgtctccc tccagagcct ccctccggacaatgagtccc ccctcttgtc 822 tcccaccctg agattgggca tggggtgcgg tgtggggggcatgtgctgcc tgttgttatg 882 ggtttttttt gcgggggggg ttgctttttt ctggggtctttgagctccaa aaaaataaac 942 acttcctttg agggagagca caccttaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaattc 1002 gggcggccgc c 1013 104 131 PRT murine Aminoacid sequence of anti-HM 1.24 antibody L chain V region 104 Met Gly PheLys Met Glu Ser His Phe Leu Val Phe Val Phe Val Phe -20 -15 -10 Leu TrpLeu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His -5 -1 1 5 LysPhe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys 10 15 20 AlaSer Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 25 30 35 40Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr 45 50 55Gly Val Pro Asp Arg Ile Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 60 65 70Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys 75 80 85Gln Gln His Tyr Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 90 95100 Glu Ile Lys 105 105 139 PRT murine Amino acid sequence of anti-HM1.24 antibody H chain V region 105 Met Glu Cys Asn Trp Ile Leu Pro PheIle Leu Ser Val Thr Ser Gly -15 -10 -5 Ala Tyr Ser Gln Val Gln Leu GlnGln Ser Gly Ala Glu Leu Ala Arg -1 1 5 10 Pro Gly Ala Ser Val Lys LeuSer Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln TrpVal Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Ile Gly Ser IlePhe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly LysAla Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln LeuSer Ile Leu Ala Phe Glu Asp Ser Ala Val 80 85 90 Tyr Tyr Cys Ala Arg GlyLeu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly ThrThr Leu Thr Val Ser Ser 110 115 120 106 126 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody L chain V region version a 106 Met Gly Trp Ser CysIle Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -15 -10 -5 Val His Ser AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala -1 1 5 10 Ser Val GlyAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val 15 20 25 Asn Thr AlaVal Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Leu LeuIle Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60 Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 Leu GlnPro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser 80 85 90 Thr ProPhe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 107 126 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody L chain V regionversion b 107 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr AlaThr Gly -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala -1 1 5 10 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys AlaSer Gln Asp Val 15 20 25 Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr ThrGly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr ThrPhe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr CysGln Gln His Tyr Ser 80 85 90 Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 95 100 105 108 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version a 108 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyArg Val Thr Met Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr Val Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 109 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version b 109 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr ValTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 110 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion c 110 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Arg Val Thr Met Thr AlaAsp Lys Ser Thr Ser 65 70 75 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 111 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version d 111 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyLys Val Thr Met Thr Ala Asp Lys Ser Thr Ser 65 70 75 Thr Val Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 112 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version e 112 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr ValTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 113 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion f 113 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr AlaAsp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 114 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version g 114 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyArg Val Thr Met Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr Val Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 115 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version h 115 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr AlaTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 116 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion i 116 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Lys Val Thr Met Thr AlaAsp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Ala PheGlu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 117 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version j 117 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyLys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 118 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version k 118 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr AlaTyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 119 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion l 119 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Lys Val Thr Met Thr AlaAsp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln Leu Ser Ile Leu Arg SerGlu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 120 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version m 120 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyLys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met GlnLeu Ser Ile Leu Arg Ser Glu Asp Ser Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 121 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version n 121 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Lys Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr AlaTyr Met Glu Leu Ser Ile Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 122 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion o 122 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Lys Val Thr Met Thr AlaAsp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg SerGlu Asp Ser Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 123 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version p 123 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyArg Val Thr Met Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr Ala Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 124 139 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of reshaped humananti-HM 1.24 antibody H chain V region version q 124 Met Asp Trp Thr TrpArg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser GlnVal Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly AlaSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpMet Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ser Ser 65 70 75 Thr ValTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 125 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V regionversion r 125 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val AlaPro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg Gln Ala ProGly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Gly Asp GlyAsp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Arg Val Thr Met Thr AlaAsp Lys Ser Thr Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Val Thr Val SerSer 110 115 120 126 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region (mouse-human hybrid) 126 Met Glu Cys Asn TrpIle Leu Pro Phe Ile Leu Ser Val Thr Ser Gly -15 -10 -5 Ala Tyr Ser GlnVal Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg -1 1 5 10 Pro Gly AlaSer Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro TyrTrp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 Glu TrpIle Gly Ser Ile Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln LysPhe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser 65 70 75 Thr ValTyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr TyrCys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 120 127 139 PRTArtificial Sequence Description of Artificial Sequence Amino acidsequence of reshaped human anti-HM 1.24 antibody H chain V region(human-mouse hybrid) 127 Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu AlaVal Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln Leu Val Gln Ser GlyAla Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys LysAla Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met Gln Trp Val Arg GlnAla Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro GlyAsp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr LeuThr Ala Asp Lys Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln Leu Ser Ile LeuAla Phe Glu Asp Ser Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg ArgGly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln Gly Thr Thr Leu ThrVal Ser Ser 110 115 120 128 139 PRT Artificial Sequence Description ofArtificial Sequence Amino acid sequence of reshaped human anti-HM 1.24antibody H chain V region version s 128 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -15 -10 -5 Ala His Ser Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys -1 1 5 10 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Pro Tyr Trp Met GlnTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly SerIle Phe Pro Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gln Lys Phe Lys GlyArg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser 65 70 75 Thr Ala Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala ArgGly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 105 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser 110 115 120 129 180 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of HM 1.24antigen 129 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu AspGly 1 5 10 15 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu ValLeu Leu 20 25 30 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr IleLys Ala 35 40 45 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met GluCys Arg 50 55 60 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu Ala GlnLys Gly 65 70 75 80 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr Cys Asn HisThr Val Met 85 90 95 Ala Leu Met Ala Ser Leu Asp Ala Glu Lys Ala Gln GlyGln Lys Lys 100 105 110 Val Glu Glu Leu Glu Gly Glu Ile Thr Thr Leu AsnHis Lys Leu Gln 115 120 125 Asp Ala Ser Ala Glu Val Glu Arg Leu Arg ArgGlu Asn Gln Val Leu 130 135 140 Ser Val Arg Ile Ala Asp Lys Lys Tyr TyrPro Ser Ser Gln Asp Ser 145 150 155 160 Ser Ser Ala Ala Ala Pro Gln LeuLeu Ile Val Leu Leu Gly Leu Ser 165 170 175 Ala Leu Leu Gln 180 130 80PRT Unknown Description of Unknown Organism HuSGI 130 Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala 20 25 30 Pro Lys LeuLeu Ile Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 35 40 45 Ser Gly ThrAsp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 50 55 60 Phe Ala ThrTyr Tyr Cys Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 65 70 75 80 131 80PRT Unknown Description of Unknown Organism REI 131 Asp Ile Gln Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val ThrIle Thr Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala 20 25 30 Pro Lys Leu LeuIle Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 35 40 45 Ser Gly Thr AspPhe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp 50 55 60 Ile Ala Thr TyrTyr Cys Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 65 70 75 80 132 44 PRTUnknown Description of Unknown Organism HuSGI 132 Glu Val Gln Leu ValGln Ser Gly Ala Asp Val Lys Lys Pro Gly Xaa 1 5 10 15 Ser Val Xaa ValSer Cys Lys Ala Ser Gly Tyr Thr Phe Ser Trp Val 20 25 30 Arg Gln Ala ProGly Xaa Gly Leu Asp Trp Val Gly 35 40 133 44 PRT Unknown Description ofUnknown Organism HG3 133 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val LysLys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly TyrThr Phe Asn Trp Val 20 25 30 Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetGly 35 40 134 32 PRT Unknown Description of Unknown Organism HuSGI 134Arg Val Thr Xaa Thr Xaa Asp Xaa Ser Xaa Asn Thr Ala Tyr Met Glu 1 5 1015 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 2530 135 32 PRT Unknown Description of Unknown Organism HG3 135 Arg ValThr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 1 5 10 15 LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 13611 PRT Unknown Description of Unknown Organism HuSGI 136 Trp Gly Gln GlyThr Leu Val Thr Val Ser Ser 1 5 10 137 11 PRT Unknown Description ofUnknown Organism JH6 137 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 510

1. A chimeric L chain comprising the constant region (C region) of ahuman light (L) chain, and the variable (V) region of the L chain of ananti-HM 1.24 antibody.
 2. The chimeric L chain according to claim 1wherein said V region of the L chain has the amino acid sequence as setforth in SEQ ID NO:
 1. 3. The chimeric L chain according to claim 1wherein said C region of the human L chain is Ck.
 4. A chimeric H chaincomprising the constant region of a human heavy(H) chain, and the Vregion of the H chain of an anti-HM 1.24 antibody.
 5. The chimeric Hchain according to claim 4 wherein said V region of the H chain has theamino acid sequence as set forth in SEQ ID NO:
 2. 6. The chimeric Hchain according to claim 4 wherein said C region of the human H chain isCγ.
 7. A Chimeric antibody comprising (1) an L chain comprising the Cregion of a human L chain and the V region of the L chain of an anti-HM1.24 antibody; and (2) an H chain comprising the C region of a human Hchain and the V region of the H chain of an anti-HM 1.24 antibody. 8.The chimeric antibody according to claim 7 wherein said V region of theL chain has the amino acid sequence as set forth in SEQ ID NO: 1, andsaid V region of the H chain has the amino acid sequence as set forth inSEQ Id no:
 2. 9. The V region of the reshaped human L chain of anti-HM1.24 antibody comprising (1) the framework region (FR) of the V regionof a human L chain, and (2) the CDR of the V region of the L chain of ananti-HM 1.24 antibody.
 10. The V region of the reshaped human L chainaccording to claim 9 wherein said CDR has the amino acid sequencerepresented by the following amino acid sequences: CDR1: Lys Ala Ser GlnAsp Val Asn Thr Ala Val Ala (SEQ ID NO: 3) CDR2: Ser Ala Ser Asn Arg TyrThr (SEQ ID NO: 4) CDR3: Gln Gln His Tyr Ser Thr Pro Phe Thr (SEQ ID NO:5).
 11. The V region of the reshaped human L chain according to claim 10wherein said FR is derived from the FR of a human antibody of humansubgroup I (HSGI).
 12. The V region of the reshaped human L chainaccording to claim 11 wherein said FR is derived from the FR of humanantibody REI.
 13. The V region of the reshaped human L chain accordingto claim 11 wherein said FR is substantially the same as the FR of humanantibody REI.
 14. The V region of the reshaped human L chain accordingto claim 11 wherein said V region of the L chain has the amino acidsequence represented as RVLa in Table
 1. 15. The V region of thereshaped human H chain of anti-HM 1.24 antibody comprising (1) the FR ofthe V region of a human H chain, and (2) the CDR of the V region of theH chain of an anti-HM 1.24 antibody.
 16. The V region of the reshapedhuman H chain according to claim 15 wherein said CDR has the amino acidsequence represented by the following amino acid sequences: CDR1: ProTyr Trp Met Gln (SEQ ID NO: 6) CDR2: Ser Ile Phe Gly Asp Gly Asp Thr ArgTyr Ser Gin Lys Phe Lys Gly (SEQ ID NO: 7) CDR3: Gly Leu Arg Arg Gly GlyTyr Tyr Phe Asp Tyr (SEQ ID NO: 8).
 17. The V region of the reshapedhuman H chain according to claim 16 wherein said FR is derived from theFR of a human antibody of HSGI.
 18. The V region of the reshaped human Hchain according to claim 16 wherein said FR 1-3 is derived from the FR1-3 of human antibody HG3.
 19. The V region of the reshaped human Hchain according to claim 16 wherein said FR 1-3 is substantially thesame as the FR 1-3 of human antibody HG3, and said FR4 is substantiallythe same the FR4 of human antibody JH6.
 20. The H chain of the reshapedhuman antibody according to claim 16 wherein the amino acid at position30 according to the definitions of Kabat in said FR1 is threonine, theamino acid at position 71 according to the definitions of Kabat in saidFR3 is alanine, and the amino acid at position 78 according to thedefinitions of Kabat in said FR3 is alanine.
 21. The H chain of thereshaped human antibody according to claim 16 wherein the amino acid atposition 73 according to the definitions of Kabat in said FR3 is lysine.22. The V region of the reshaped human H chain according to claim 17wherein said V region of the H chain has the amino acid sequencerepresented as RVHf, RVHh, RVHi, RVHj, RVHk, RVHl, RVHfm, RVHn, RVHO,RVHp, RVHr, or RVHs in Table 2 to
 4. 23. The reshaped human L chain ofanti-HM 1.24 antibody comprising (1) the C region of a human L chain,and (2) the V region of an L chain comprising the FR of a human L chainand the CDR of the L chain of an anti-HM 1.24 antibody.
 24. The reshapedhuman L chain according to claim 23 wherein said C region of the human Lchain is human CK region, said FR of the human L chain is derived fromthe FR of a human antibody of HSGI, and said CDR of then L chain has theamino acid sequence shown in claim
 10. 25. The V region of the reshapedhuman L chain according to claim 23 wherein said FR is derived from theFR of human antibody REI.
 26. The V region of the reshaped human L chainaccording to claim 23 wherein said FR is substantially the same as theFR of human antibody REI.
 27. The reshaped human L chain according toclaim 23 wherein said V region of the L chain has the amino acidsequence represented as RVLa in Table
 3. 28. The reshaped human H chainof anti-HM 1.24 antibody comprising (1) the C region of a human H chain,and (2) the V region of an H chain comprising the FR of a human H chainand the CDR of the H chain of an anti-HM 1.24 antibody.
 29. The reshapedhuman H chain according to claim 28 wherein said C region of the human Hchain is human Cγ1 region, said FR of the human H chain is derived fromthe FR of the human antibody of HSGI, and said CDR of the H chain hasthe amino acid sequence shown in claim
 16. 30. The reshaped human Hchain according to claim 28 wherein said FR 1-3is derived from the FR1-3 of human antibody HG3 and said FR4 is derived from the FR4 of humanantibody JH6.
 31. The reshaped human H chain according to claim 28wherein said FR 1-3is substantially the same the FR 1-3 of humanantibody HG3 and said FR4 is substantially the same the FR4 of humanantibody JH6.
 32. The H chain of the reshaped human antibody accordingto claim 28 wherein the amino acid at position 30 according to thedefinitions of Kabat in said FR1 is threonine, the amino acid atposition 71 according to the definitions of Kabat in said FR3 isalanine, and the amino acid at position 78 according to the definitionsof Kabat in said FR3 is alanine.
 33. The H chain of the reshaped humanantibody according to claim 28 wherein the amino acid at position 73according to the definitions of Kabat in said FR3 is lysine.
 34. Thereshaped human H chain according to claim 28 wherein said V region ofthe H chain has the amino acid sequence represented as RVHf, RVHh, RVHi,RVHj, RVHk, RVHl, RVHfm, RVHn, RVHO, RVHp, RVHr, or RVHs in Table 2 to4.
 35. The reshaped human antibody of anti-HM 1.24 antibody comprising:(A) an L chain comprising (1) the C region of a human L chain, and (2)the V region of an L chain comprising the FR of a human L chain and theCDR of the L chain of an anti-HM 1.24 antibody; and (B) an H chaincomprising (1) the C region of a human H chain, and (2) the V region ofan H chain comprising the FR of a human H chain and the CDR of the Hchain of an anti-HM 1.24 antibody.
 36. The reshaped human antibodyaccording to claim 35 wherein said CDR of the L chain has the amino acidsequence shown in claim 10 and said CDR of the H chain has the aminoacid sequence shown in claim
 16. 37. The reshaped human antibodyaccording to claim 35 wherein said CDR of the L chain has the amino acidsequence shown in claim 10; said CDR of the H chain has the amino acidsequence shown in claim 16; said FR of a human L chain is derived fromthe FR of an antibody of HSGI; said FR of the human H chain id derivedfrom the FR of a human antibody of HSGI; said C region of the human Lchain is human Ck region; and, said C region of the human H chain ishuman Cγ1 region.
 38. The reshaped human antibody according to claim 35wherein said FR of the L chain is derived from the FR of human antibodyREI, said FR 1-3 of the H chain is derived from human antibody HG3, andsaid FR4 of the H chain is derived from the FR4 of human antibody JH6.39. The reshaped human antibody according to claim 35 wherein said Vregion of the L chain has the amino acid sequence represented as RVLa inTable
 1. 40. The reshaped human antibody according to claim 35 whereinsaid V region of the H chain has the amino acid sequence represented asRVHf, RVHh, RVHI, RVHj, RVHk, RVHl, RVHfm, RVHn, RVHO, RVHp, RVHr, orRVHs in Table 2 to
 4. 41. DNA encoding the V region of the L chain of ananti-HM 1.24 antibody.
 42. The DNA according to claim 41 wherein said Vregion of the L chain encodes the amino acid sequence as set forth inSEQ ID NO:
 1. 43. The DNA according to claim 41 wherein DNA that encodessaid V region of the L chain has nucleotide sequence as set forth in SEQID NO:
 1. 44. DNA encoding the V region of the H chain of an anti-HM1.24 antibody.
 45. The DNA according to claim 44 wherein said V regionof the H chain encodes the amino acid sequence as set forth in SEQ IDNO:
 2. 46. The DNA according to claim 44 wherein DNA that encodes said Vregion of the H chain has the nucleotide sequence as set forth in SEQ IDNO:
 2. 47. DNA encoding a chimeric L chain comprising: (1) the C regionof a human L chain; and (2) the V region of the L chain of an anti-HM1.24 antibody.
 48. The DNA according to claim 47 wherein said V regionof the L chain encodes the amino acid sequence as set forth in SEQ IDNO:
 1. 49. The DNA according to claim 47 wherein said V region of the Lchain has the nucleotide sequence as set forth in SEQ ID NO:
 1. 50. DNAencoding a chimeric H chain comprising: (1) the C region of a human Hchain; and (2) the V region of the H chain of an anti-HM 1.24 antibody.51. The DNA according to claim 50 wherein said V region of the H chainencodes the amino acid sequence as set forth in SEQ ID NO:
 2. 52. TheDNA according to claim 50 wherein said V region of the H chain has thenucleotide sequence as set forth in SEQ ID NO:
 2. 53. DNA encoding the Vregion of the reshaped human L chain of an anti-HM 1.24 antibodycomprising: (1) the FR of the V region of a human L chain; and (2) theCDR of the V region of the L chain of an anti-HM 1.24 antibody.
 54. DNAencoding the V region of the reshaped human L chain according to claim53 wherein said CDR has the amino acid sequence shown in claim
 10. 55.DNA encoding the V region of the reshaped human L chain according toclaim 53 wherein said FR is derived from the FR of the human antibody ofHSGI.
 56. DNA encoding the V region of the reshaped human L chainaccording to claim 53 wherein said FR is derived from the FR of humanantibody REI.
 57. DNA encoding the V region of the reshaped human Lchain according to claim 53 wherein said FR is substantially the samethe FR of human antibody REI.
 58. DNA according to claim 51 wherein saidV region of the L chain encodes the amino acid sequence represented asRVLa in Table
 1. 59. DNA encoding the V region of the reshaped human Lchain according to claim 53, which has the nucleotide sequence as setforth in SEQ ID NO:
 9. 60. DNA encoding the V region of the reshapedhuman H chain of an anti-HM 1.24 antibody comprising: (1) the FR of theV region of a human H chain; and (2) the CDR of the V region of the Hchain of an anti-HM 1.24 antibody.
 61. DNA encoding the V region of thereshaped human H chain according to claim 60 wherein said CDR has theamino acid sequence shown in claim
 16. 62. DNA encoding the V region ofthe reshaped human H chain according to claim 60 wherein said FR isderived from the FR of the human antibody of HSGI.
 63. DNA encoding theV region of the reshaped human H chain according to claim 60 whereinsaid FR 1-3 is derived from the FR 1-3 of human antibody HG3.
 64. DNAencoding the V region of the reshaped human H chain according to claim60 wherein said FR 1-3 is substantially the same as the FR 1-3 of humanantibody HG3.
 65. DNA encoding the V region of the H chain of thereshaped human antibody according to claim 60 wherein the amino acid atposition 30 according to the definitions of Kabat in said FR1 isthreonine, the amino acid at position 71 according to the definitions ofKabat in said FR3 is alanine, and the amino acid at position 78according to the definitions of Kabat in said FR3 is alanine.
 66. DNAencoding the V region of the H chain of the reshaped human antibodyaccording to claim 60 wherein the amino acid at position 73 according tothe definitions of Kabat in said FR3 is lysine.
 67. DNA encoding the Vregion of the reshaped human H chain according to claim 60 wherein saidV region of the H chain has the amino acid sequence represented as RVHf,RVHh, RVHi, RVHj, RVHk, RVHl, RVHfm, RVHn, RVHO, RVHP, RVHr, or RVHs inTable 2 to
 4. 68. DNA encoding the V region of the reshaped human Hchain according to claim 60, which has the nucleotide sequence as setforth in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, or
 102. 69.DNA encoding the reshaped human L chain of an anti-HM 1.24 antibodycomprising: (1) the C region of a human L chain; and (2) the V region ofan L chain comprising the FR of a human L chain and the CDR of the Lchain of an anti-HM 1.24 antibody.
 70. DNA according to claim 69 whereinsaid V region of the L chain encodes the amino acid sequence representedas RVLa in Table
 1. 71. DNA according to claim 69 wherein said V regionof the L chain has the nucleotide sequence as set forth in SEQ ID NO: 9.72. DNA according to claim 69 wherein said C region of the L chain isthe Ck region of a human L chain.
 73. DNA encoding the reshaped human Hchain of an anti-HM 1.24 antibody comprising: (1) the C region of ahuman H chain; and (2) the V region of an H chain comprising the FR of ahuman H chain and the CDR of the H chain of an anti-HM 1.24 antibody.74. DNA encoding the reshaped human H chain according to claim 73wherein said V region of the H chain has the amino acid sequencerepresented as RVHf, RVHh, RVHi, RVHj, RVHk, RVHl, RVHfm, RVHn, RVHO,RVHp, RVHr, or RVHs in Table 2 to
 4. 75. DNA encoding the reshaped humanH chain according to claim 73 wherein said V region of the H chain hasthe nucleotide sequence as set forth in SEQ ID NO: 18, 19, 20, 21, 22,23, 24, 25, 26, 28, or
 102. 76. DNA encoding. the reshaped human H chainaccording to claim 73 wherein said C region of the human H chain is theCγ1 region of the human H chain.
 77. A vector comprising the DNAaccording to any of claims 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, and
 76. 78. A host cell transformed with a vectorcomprising the DNA according to any of claims 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 50, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and
 76. 79. A method forproducing the chimeric antibody of anti-HM 1.24 antibody, comprising thesteps of: culturing a host cell co-transformed with an expression vectorcomprising the DNA according to any of claims 41, 42, 43, 47, 48, and49, and an expression vector comprising the DNA according to any ofclaims 44, 45, 46, 50 ,51, and 52; and recovering the desired antibody.80. A method for producing the reshaped human antibody of anti-HM 1.24antibody, comprising the steps of: culturing a host cell co-transformedwith an expression vector comprising the DNA according to any of claims53, 54, 55, 56, 57, 58, 59, 69, 70 ,71, and 72 and an expression vectorcomprising the DNA according to any of claims 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, and 76; and recovering the desiredantibody.
 81. A pharmaceutical composition containing as an activeingredient a chimeric antibody which specifically recognizes polypeptidehaving the amino acid sequence as set forth in SEQ ID NO:
 103. 82. Apharmaceutical composition containing as an active ingredient chimericanti-HM 1.24 antibody.
 83. A therapeutic agent for myeloma containing asan active ingredient a chimeric antibody which recognizes polypeptidehaving the amino acid sequence as set forth in SEQ ID NO:
 103. 84. Atherapeutic agent for myeloma containing as an active ingredientchimeric anti-HM 1.24 antibody.
 85. A pharmaceutical compositioncontaining as an active ingredient a reshaped human antibody whichspecifically recognizes polypeptide having the amino-acid sequence asset forth in SEQ ID NO:
 103. 86. A pharmaceutical composition containingas an active ingredient reshaped human anti-HM 1.24 antibody.
 87. Atherapeutic agent for myeloma containing as an active ingredient areshaped human antibody which recognizes polypeptide having the aminoacid sequence as set forth in SEQ ID NO:
 103. 88. A therapeutic agentfor myeloma containing as an active ingredient reshaped human anti-HM1.24 antibody.